Compositions for linking DNA-binding domains and cleavage domains

ABSTRACT

Disclosed herein are compositions for linking DNA binding domains and cleavage domains (or cleavage half-domains) to form non-naturally occurring nucleases. Also described are methods of making and using compositions comprising these linkers.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/471,782 filed Aug. 28, 2014 which claims the benefit of U.S.Provisional Application No. 61/871,219, filed Aug. 28, 2013, thedisclosures of which are hereby incorporated by reference in theirentireties.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 5, 2017, isnamed 8325010801SL.txt and is 92,111 bytes in size.

TECHNICAL FIELD

The present disclosure is in the fields of genome and proteinengineering.

BACKGROUND

Artificial nucleases, such as engineered zinc finger nucleases (ZFN),transcription-activator like effector nucleases (TALENs), the CRISPR/Cassystem with an engineered crRNA/tracr RNA (single guide RNA′) and/ornucleases based on the Argonaute system (e.g., from T. thermophilus,known as ‘TtAgo’, (Swarts et al (2014) Nature 507(7491): 258-261),comprising DNA binding domains (nucleotide or polypeptide) operablylinked to cleavage domains have been used for targeted alteration ofgenomic sequences. For example, zinc finger nucleases have been used toinsert exogenous sequences, inactivate one or more endogenous genes,create organisms (e.g., crops) and cell lines with altered geneexpression patterns, and the like. See, e.g., U.S. Pat. Nos. 8,586,526;8,329,986; 8,399,218; 6,534,261; 6,599,692; 6,503,717; 6,689,558;7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796; 7,951,925;8,110,379; 8,409,861; U.S. Patent Publications 20030232410; 20050208489;20050026157; 20050064474; 20060063231; 20080159996; 201000218264;20120017290; 20110265198; 20130137104; 20130122591; 20130177983;20130177960 and 20150056705, the disclosures of which are incorporatedby reference in their entireties for all purposes. For instance, a pairof nucleases (e.g., zinc finger nucleases, TALENs) is typically used tocleave genomic sequences. Each member of the pair generally includes anengineered (non-naturally occurring) DNA-binding protein linked to oneor more cleavage domains (or half-domains) of a nuclease. When theDNA-binding proteins bind to their target sites, the cleavage domainsthat are linked to those DNA binding proteins are positioned such thatdimerization and subsequent cleavage of the genome can occur, generallybetween the pair of the zinc finger nucleases or TALENs.

It has been shown that cleavage activity of the nuclease pair is relatedto the length of the linker joining the zinc finger and the cleavagedomain (“ZC” linker), the amino acid composition, and the distancebetween the target sites (binding sites). See, for example, U.S. Pat.Nos. 8,772,453; 7,888,121 and 8,409,861; Smith et al. (2000) NucleicAcids Res. 28:3361-3369; Bibikova et al. (2001) Mol. Cell. Biol.21:289-297. When using pairs of nuclease fusion proteins, optimalcleavage with currently available ZC linkers and cleavage half domainshas been obtained when the binding sites for the fusion proteins arelocated 5 or 6 nucleotides apart (as measured from the near edge of eachbinding site). See, e.g., U.S. Pat. No. 7,888,121. U.S. PatentPublication 20090305419 describes linking DNA-binding domains andcleavage domains by using a ZC linker and modifying the N-terminalresidues of the FokI cleavage domain.

Thus, there remains a need for methods and compositions that allowtargeted modification where the artificial nucleases can cleaveendogenous genomic sequences with binding site separations other than 5bp or 6 bp. The ability to target sequences with different spacingswould increase the number of genomic targets that can be cleaved.Altering the preferences between target sites separated by differentnumbers of base pairs could also allow the artificial nucleases to actwith greater specificity.

SUMMARY

Disclosed herein are compositions for linking DNA-binding domains andcleavage domains to form nucleases, for example nucleases with alteredtarget site separation (gap) preferences as compared to conventionallinkers. Also described are fusion proteins comprising these linkers.The disclosure also provides methods of using these fusion proteins andcompositions thereof for targeted cleavage of cellular chromatin in aregion of interest and/or homologous recombination at a predeterminedregion of interest in cells.

Thus, in one aspect, described herein are amino acid sequences linking aDNA-binding domain (e.g., zinc finger protein or TAL-effector domain) toa cleavage domain (e.g., wild type or engineered FokI cleavage domain).In certain embodiments, the amino acid linker sequences extend betweenthe last residue of the N- or C-terminal of the DNA-binding domain andthe N- or C-terminal of the cleavage domain. In other embodiments, thelinker may replace one or more residues of the DNA-binding domain and/orcleavage domain. In certain embodiments, the linker is 8, 9, 10, 11, 12,13, 14, 15, 16, 17 or more residues in length. In other embodiments, thelinker between the DNA-binding domain and the cleavage domain (orcleavage half-domain) comprises any of the linkers shown in FIGS. 4, 7,9, 11, 12, 13, 15 and 16 (SEQ ID NOs: 2-21, 45-49, 51, 62-226, 233-240,and 258-312) (with or without 1 or more of the N-terminal amino acidresidues shown in these Figures), including but not limited to(N)GICPPPRPRTSPP (SEQ ID NO:2); (T)GTAPIEIPPEVYP (SEQ ID NO:3);(N)GSYAPMPPLALASP (SEQ ID NO:4); (P)GIYTAPTSRPTVPP (SEQ ID NO:5);(N)GSQTPKRFQPTHPSA (SEQ ID NO:6); (H)LPKPANPFPLD (SEQ ID NO:7);(H)RDGPRNLPPTSPP (SEQ ID NO:8); (H)RLPDSPTALAPDTL (SEQ ID NO:9);(DPNS)PISRARPLNPHP (SEQ ID NO:10); (Y)GPRPTPRLRCPIDSLIFR (SEQ ID NO:11);(H)CPASRPIHP (SEQ ID NO:12); (G)LQSLIPQQLL (SEQ ID NO:13);(G)LQPTVNHEYNN (SEQ ID NO:14); (P)ANIHSLSSPPPL (SEQ ID NO:15);(P)AGLNTPCSPRSRSN (SEQ ID NO:16); (A)TITDPNP (SEQ ID NO:17); (P)PHKGLLP(SEQ ID NO:18); (S)VSLPDTHH (SEQ ID NO:19); (G)THGATPTHSP (SEQ IDNO:20); (V)APGESSMTSL (SEQ ID NO:21), (LRGS)PISRARPLNPHP (SEQ ID NO:22),(LRGS)ISRARPLNPHP (SEQ ID NO:23), (LRGS)PSRARPLNPHP (SEQ ID NO:24),(LRGS)SRARPLNPHP (SEQ ID NO:25), (LRGS)YAPMPPLALASP (SEQ ID NO:26),(LRGS)APMPPLALASP (SEQ ID NO:27), (LRGS)PMPPLALASP (SEQ ID NO:28),(LRGS)MPPLALASP (SEQ ID NO:29) and HLPKPANPFPLD (SEQ ID NO:30), whereinthe amino acid residue(s) shown in round parentheses is(are) optionallypresent. In certain embodiments, the linker comprises a sequenceselected from the group consisting of PKPAN (SEQ ID NO:31), RARPLN (SEQID NO:32); PMPPLA (SEQ ID NO:33) or PPPRP (SEQ ID NO:34). In certainembodiments, the linkers as described herein further comprise a ZClinker sequence at their N-terminal ends. In other embodiments, thelinker is includes modifications at the junction with the cleavagedomain (FokI), for example as shown in FIGS. 4, 7, 9, 11, 12, 13, 15 and16.

In still further aspects, described herein is a fusion proteincomprising a DNA-binding domain, a modified FokI cleavage domain and aZC linker between the DNA-binding domain and the FokI cleavage domain.The FokI cleavage domain may be modified in any way, including addition,deletion and/or substitution of one or more amino acids residues. Incertain embodiments, the modifications to the FokI cleavage domaincomprises one or more additions, deletions and/or substitutions to theN-terminal region of FokI (residues 158-169 of SEQ ID NO:1), including,for example, addition, deletion and/or substitution of 1, 2, 3, 4 aminoacid residues. In certain embodiments, the modified FokI cleavage domaincomprises deletions of 1, 2, 3, 4 or more amino acids from theN-terminal region of FokI (e.g., deletion of one or more of residues158, 159, 160 and/or 161 of the wild-type FokI domain of SEQ ID NO:1).In other embodiments, the modified FokI cleavage domain comprises one ormore deletions from, and one or more substitutions within, theN-terminal region of FokI (e.g., deletion of one or more of residues158-161 and substitution of one or more of the remaining residues). Inother embodiments, the modified FokI cleavage domain comprises one ormore substitutions in the N-terminal region of FokI. In still furtherembodiments, the modified FokI cleavage domain comprises one or moreadditional amino acid residues (e.g., 1, 2, 3, 4 or more)N-terminal tothe N-terminal-most residue of FokI (residue 158 of SEQ ID NO:1). Inother embodiments, the modified FokI cleavage domain comprises one ormore additional amino acid residues (e.g., 1, 2, 3, 4 or more)N-terminalto the N-terminal-most residue of FokI (residue 158 of SEQ ID NO:1)and/or one or more substitutions within the N-terminal region of FokI.In certain embodiments, the fusion protein comprises a modified FokIcleavage domain as shown in FIG. 15 or FIG. 16. In other embodiments,the fusion protein comprises a modified FokI cleavage domain as shown inFIG. 15 or FIG. 16 and further comprising one or more modificationswithin the N-terminal as shown in U.S. Patent Publication No.20090305419.

In another aspect, described herein is a dimer comprising at least twofusion proteins, each fusion protein comprising a DNA-binding domain,linker and cleavage domain. In certain embodiments, at least one fusionprotein comprises a linker as described herein. In other embodiments,both fusion proteins comprise a linker as described herein. In stillfurther embodiments, the DNA-binding domains of the dimer targetsequences (e.g., in double-stranded DNA) separated by 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16 (or more) base pairs, preferably 5 to 8 basepairs. Any of the fusion proteins may comprise wild-type or engineeredcleavage domains.

In another aspect, fusion polypeptides comprising a DNA-binding domain(e.g., a zinc finger or TALE DNA-binding domain), a cleavage half-domainand a linker as described herein are provided.

In another aspect, polynucleotides encoding any of the linkers or fusionproteins as described herein are provided. In some embodiments, thepolynucleotides are RNAs.

In yet another aspect, cells comprising any of the polypeptides (e.g.,fusion polypeptides) and/or polynucleotides as described herein are alsoprovided. In one embodiment, the cells comprise a pair of fusionpolypeptides, each comprising a cleavage domain as disclosed herein.

In yet another aspect, methods for targeted cleavage of cellularchromatin in a region of interest; methods of causing homologousrecombination to occur in a cell; methods of treating infection; and/ormethods of treating disease are provided. The methods involve cleavingcellular chromatin at a predetermined region of interest in cells byexpressing a pair of fusion polypeptides, at least one of whichcomprises a linker as described herein. In certain embodiments, onefusion polypeptide comprises a linker as described herein and in otherembodiments, both fusion polypeptides comprise a linker as describedherein. Furthermore, in any of the methods described herein, the pair offusion polypeptides cleaves the targeted region when the binding sitesfor the zinc finger nucleases are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16 or even more base pairs apart.

The polypeptides comprising the linkers as described herein can be usedin methods for targeted cleavage of cellular chromatin in a region ofinterest and/or homologous recombination at a predetermined region ofinterest in cells. Cells include cultured cells, cells in an organismand cells that have been removed from an organism for treatment in caseswhere the cells and/or their descendants will be returned to theorganism after treatment. A region of interest in cellular chromatin canbe, for example, a genomic sequence or portion thereof.

A fusion protein can be expressed in a cell, e.g., by delivering thefusion protein to the cell or by delivering a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide, if DNA, istranscribed, and an RNA molecule delivered to the cell or a transcriptof a DNA molecule delivered to the cell is translated, to generate thefusion protein. Methods for polynucleotide and polypeptide delivery tocells are presented elsewhere in this disclosure.

Accordingly, in another aspect, a method for cleaving cellular chromatinin a region of interest can comprise (a) selecting a first sequence inthe region of interest; (b) engineering a first DNA-binding domain tobind to the first sequence; (c) expressing a first fusion protein in thecell, the first fusion protein comprising the first DNA-binding domain(e.g., zinc finger or TALE), and a cleavage half-domain; and (d)expressing a second fusion protein in the cell, the second fusionprotein comprising a second DNA-binding domain, and a second cleavagehalf-domain, wherein at least one of the fusion proteins comprises alinker as described herein, and further wherein the first fusion proteinbinds to the first sequence, and the second fusion protein binds to asecond sequence located between 2 and 50 nucleotides from the firstsequence, such that cellular chromatin is cleaved in the region ofinterest. In certain embodiments, both fusion proteins comprise a linkeras described herein.

In other embodiments, the disclosure provides methods of cleavingcellular chromatin by introducing one more nucleases comprising a linkeras described herein into a cell such that the nucleases target andcleave the cellular chromatin of the cell. The nuclease may comprise azinc finger nuclease (ZFN), a TALE-nuclease (TALEN), TtAgo or aCRISPR/Cas nuclease system or a combination thereof. The nuclease(s) maybe introduced into the cell in any form, for example in protein form, inmRNA form or carried on a viral (AAV, IDLV, etc.) vector or non-viralvector (e.g., plasmid). In certain embodiments, the methods comprise (a)selecting first and second sequences in a region of interest, whereinthe first and second sequences are between 2 and 50 nucleotides apart;(b) engineering a first DNA-binding domain to bind to the firstsequence; (c) engineering a second DNA-binding domain to bind to thesecond sequence; (d) expressing a first fusion protein in the cell, thefirst fusion protein comprising the first engineered DNA-binding domain,a first linker as described herein, and a first cleavage half domain;(e) expressing a second fusion protein in the cell, the second fusionprotein comprising the second engineered DNA-binding domain, a secondlinker and a second cleavage half-domain; wherein the first fusionprotein binds to the first sequence and the second fusion protein bindsto the second sequence, thereby cleaving the cellular chromatin in theregion of interest. In certain embodiments, the first and second fusionproteins comprise a linker as described herein.

Also provided are methods of altering a region of cellular chromatin,for example to introduce targeted mutations. In certain embodiments,methods of altering cellular chromatin comprise introducing into thecell one or more targeted nucleases to create a double-stranded break incellular chromatin at a predetermined site, and a donor polynucleotide,having homology to the nucleotide sequence of the cellular chromatin inthe region of the break. Cellular DNA repair processes are activated bythe presence of the double-stranded break and the donor polynucleotideis used as a template for repair of the break, resulting in theintroduction of all or part of the nucleotide sequence of the donor intothe cellular chromatin. Thus, a sequence in cellular chromatin can bealtered and, in certain embodiments, can be converted into a sequencepresent in a donor polynucleotide.

Targeted alterations include, but are not limited to, point mutations(i.e., conversion of a single base pair to a different base pair),substitutions (i.e., conversion of a plurality of base pairs to adifferent sequence of identical length), insertions or one or more basepairs, deletions of one or more base pairs and any combination of theaforementioned sequence alterations.

The donor polynucleotide can be DNA or RNA, can be linear or circular,and can be single-stranded or double-stranded. It can be delivered tothe cell as naked nucleic acid, as a complex with one or more deliveryagents (e.g., liposomes, poloxamers) or contained in a viral deliveryvehicle, such as, for example, an adenovirus or an adeno-associatedVirus (AAV). Donor sequences can range in length from 10 to 1,000nucleotides (or any integral value of nucleotides therebetween) orlonger. In some embodiments, the donor comprises a full length geneflanked by regions of homology with the targeted cleavage site. In someembodiments, the donor lacks homologous regions and is integrated into atarget locus through homology independent mechanism (i.e. NHEJ). Inother embodiments, the donor comprises a smaller piece of nucleic acidflanked by homologous regions for use in the cell (i.e. for genecorrection). In some embodiments, the donor comprises a gene encoding afunctional or structural component such as a shRNA, RNAi, miRNA or thelike. In other embodiments the donor comprises sequences encoding aregulatory element that binds to and/or modulates expression of a geneof interest. In other embodiments, the donor is a regulatory protein ofinterest (e.g. ZFP TFs, TALE TFs or a CRISPR/Cas TF) that binds toand/or modulates expression of a gene of interest.

In certain embodiments, the frequency of homologous recombination can beenhanced by arresting the cells in the G2 phase of the cell cycle and/orby activating the expression of one or more molecules (protein, RNA)involved in homologous recombination and/or by inhibiting the expressionor activity of proteins involved in non-homologous end-joining.

In any of the methods described herein in which a pair of nucleases isused, the first and second nucleases of the nuclease pair can bind totarget sites 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 or more base pairs apart. In addition, in any of the methods, thesecond zinc finger binding domain may be engineered to bind to thesecond sequence.

Furthermore, in any of the methods described herein, the fusion proteinsmay be encoded by a single polynucleotide.

For any of the aforementioned methods, the cellular chromatin can be ina chromosome, episome or organellar genome. Cellular chromatin can bepresent in any type of cell including, but not limited to, prokaryoticand eukaryotic cells, fungal cells, plant cells, animal cells, mammaliancells, primate cells and human cells.

In another aspect, described herein is a kit comprising a linker asdescribed herein or a polynucleotide encoding a linker as describedherein; ancillary reagents; and optionally instructions and suitablecontainers. The kit may also include one or more nucleases orpolynucleotides encoding such nucleases.

In any of the proteins, methods and kits described herein, the cleavagedomain (or cleavage half-domain) may comprise a TypeIIS cleavage domain,such as a cleavage half-domain from FokI.

These and other aspects will be readily apparent to the skilled artisanin light of disclosure as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the sequence of an exemplary zinc finger nuclease thatbinds to a target site in CCR5 (SEQ ID NO:1). The zinc finger DNAbinding domain is doubly underlined. The entire FokI cleavage domain isunderlined and the N-terminal region is underlined and bolded. A “ZC”linker (LRGS; SEQ ID NO:35) is shown in plain text between the zincfinger and cleavage domains.

FIGS. 2A and 2B, depict library design and selection. FIG. 2A shows hostZFN used in the bacterial selection assay (Example 3, SEQ ID NOs:52-55,respectively, in order of appearance), indicating the location of therandomized linker library. The zinc finger protein domain used forselections included the recognition helices regions of ZFP 8196 asdepicted in U.S. Pat. No. 7,951,925, which binds to the CCR5 gene. FIG.2B is a schematic showing an overview of the bacterial system used forselection of linkers.

FIG. 3 shows an overview of the target sequences (including indicatedspacings between target sites) used in the bacterial ccdB toxin plasmidof the bacterial selection system (SEQ ID NOs:56-61, respectively, inorder of appearance).

FIG. 4 depicts exemplary linkers obtained from bacterial selectionsusing libraries of 8 to 17 amino acid long linkers for the indicatedtarget spacings (SEQ ID NOs:62-226, respectively, in order of appearance(top to bottom, left to right)). “×2” and “×7” indicate the number oftimes the sequence was found in the screen.

FIG. 5 shows the distribution of the linker length (8 to 17 amino acids)relative to target spacing (5 to 16 base pairs) after selection.

FIG. 6 depicts an overview of the target sites for the modified CCR5locus in mammalian cells, including target sequences and spacings (SEQID NOs:227-232, respectively, in order of appearance).

FIG. 7 depicts results of ZFN cleavage with an 8 base pair gap betweentarget sites using the indicated linkers (SEQ ID NOs:233, 2-6, 234, 161and 235, respectively, in order of appearance).

FIG. 8 is a summary of results obtained with all linkers tested for theindicated target site gaps.

FIGS. 9A through 9C, show ZFN activity and dimer gap preference results.FIGS. 9A and 9B show the results of ZFN-modifications (“indels”)obtained with the indicated linkers (SEQ ID NOs:233, 2-6, 234, 161, 235,233, 7-11, 236, 12-16, 115, 237, 17-21, 238-240 and 71, respectively, inorder of appearance) at the indicated gaps between the paired targetsites. FIG. 9C shows dimer gap preferences of the indicated linkers.

FIGS. 10A and 10B, shows the design, assembly scheme, and results ofportability studies conducted with various linkers. FIG. 10A shows thedesign of the vectors used, including nucleotide sequences (SEQ IDNOs:241, 243, 245, 247, 249, 251, 253, 257 and 255, respectively, inorder of appearance) and amino acid sequences (SEQ ID NOs:242, 244, 246,248, 250, 252, 254, 256 and 256, respectively, in order of appearance).Solid blocks labeled ‘A’, ‘B’ and ‘C′’ indicate zinc finger modules.FIG. 10B depicts a summary of the results obtained.

FIGS. 11A and 11B, show the activity of linkers) selected to recognizean 8 or 9 bp gap between the target sites. FIG. 11A depicts gels showingthe Cel-I results for 8 pairs of ZFNs modified by 4 different linkers.Lanes are numbered according to the Group shown at the bottom of thefigure. GFP indicates the GFP expression vector control. An ‘X’ over thelane indicates a faulty run. FIG. 11B indicates the percent NHEJactivity from the Cel 1 gels in FIG. 11A. FIGS. 11A and B disclose SEQID NOS 45 and 45-49, respectively, in order of appearance.

FIG. 12 shows activity, as determined by Cell assay and sequenceanalysis, of the indicated linkers in the indicated zinc finger andcleavage domain vectors (SEQ ID NOs:258-261, 258-261 and 262-270,respectively, in order of appearance). As shown the linker sequenceextends from the amino acid residue immediately C-terminal to the 2^(nd)histidine residue of the ZFP sequence to the amino acid residueimmediately N-terminal to the EL FokI sequence. Arrows indicate the twomost active 7 bp gap linker sequences.

FIG. 13 shows activity of the indicated shows activity (% NHEJ) of theindicated linkers in the indicated zinc finger and cleavage domainvectors (SEQ ID NOs:271-279, respectively, in order of appearance). Asshown the linker sequence extends from the amino acid residueimmediately C-terminal to the 2^(nd) histidine residue of the ZFPsequence to the amino acid residue immediately N-terminal to the EL FokIsequence.

FIG. 14 is a schematic illustrating dimerization (e.g.,heterodimerization) of FokI cleavage domains upon binding of ZFNs totheir target sites with the indicated gaps.

FIG. 15 shows activity (% NHEJ) of ZFNs including linkers as describedherein and including modified junctions as between the linker andcleavage domain (in the left and/or right ZFNs of the pair as indicated)(SEQ ID NOs:280-303 and 51, respectively, in order of appearance).

FIG. 16 shows activity (% NHEJ) of different ZFNs including linkers asdescribed herein and including modified junctions as between the linkerand cleavage domain (in the left and/or right ZFNs of the pair asindicated). The linker portion of the sequences extends from the aminoacid residue immediately C-terminal to the 2nd histidine residue of theZFP sequence to the amino acid residue immediately N-terminal to the ELFokI sequence. The activities of the ZFN pairs with both conventionaljunction sequences are boxed. FIG. 16 discloses “GSKS,” “GSVKS,”“GSEVKS,” “GSQSVKS,” “GSKQLVKS,” “GSGKQLVKS,” “GSVTKQLVKS,” “GSQLVKS”and “GSTKQLVKS” as SEQ ID NOS 304-312, respectively.

DETAILED DESCRIPTION

Described herein are compositions for linking DNA-binding domains andcleavage domains to form artificial nucleases and methods of using thesenucleases for targeted alteration of a cellular nucleotide sequence,e.g., by targeted cleavage followed by non-homologous end joining; bytargeted cleavage followed by homologous recombination between anexogenous polynucleotide (comprising one or more regions of homologywith the cellular nucleotide sequence) and a genomic sequence; bytargeted inactivation of one or more endogenous genes.

Exemplary linkers as shown FIGS. 4 and 9 increase the ability of a pairof ZFNs to cleave when the ZFN target sites are more than 5 or 6 basepairs apart. Thus, certain linkers described herein significantlyincrease the ability to perform targeted genomic alteration byincreasing the cleavage activity when the zinc finger target sites arenot separated by 5 or 6 base pairs.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

Definitions

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one ormore TALE repeat domains/units. The repeat domains are involved inbinding of the TALE to its cognate target DNA sequence. A single “repeatunit” (also referred to as a “repeat”) is typically 33-35 amino acids inlength and exhibits at least some sequence homology with other TALErepeat sequences within a naturally occurring TALE protein.

Zinc finger and TALE binding domains can be “engineered” to bind to apredetermined nucleotide sequence, for example via engineering (alteringone or more amino acids) of the recognition helix region of a naturallyoccurring zinc finger or TALE protein. Therefore, engineered DNA bindingproteins (zinc fingers or TALEs) are proteins that are non-naturallyoccurring. Non-limiting examples of methods for engineering DNA-bindingproteins are design and selection. A designed DNA binding protein is aprotein not occurring in nature whose design/composition resultsprincipally from rational criteria. Rational criteria for design includeapplication of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPand/or TALE designs and binding data. See, for example, U.S. Pat. Nos.8,586,526; 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

A “selected” zinc finger protein or TALE is a protein not found innature whose production results primarily from an empirical process suchas phage display, interaction trap or hybrid selection. See e.g., U.S.Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; and 6,200,759; WO95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO01/60970 WO 01/88197, WO 02/099084 and U.S. Publication No. 20110301073.

“TtAgo” is a prokaryotic Argonaute protein thought to be involved ingene silencing. TtAgo is derived from the bacteria Thermus thermophilus.See, e.g. Swarts et al, ibid, G. Sheng et al., (2013) Proc. Natl. Acad.Sci. U.S.A. 111, 652). A “TtAgo system” is all the components requiredincluding, for example, guide DNAs for cleavage by a TtAgo enzyme.

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;” “+ and −cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Pat. Nos. 8,623,618; 7,888,121; 7,914,796; and 8,034,598 and U.S.Publication No. 20110201055, incorporated herein by reference in theirentireties.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

A “homologous, non-identical sequence” refers to a first sequence whichshares a degree of sequence identity with a second sequence, but whosesequence is not identical to that of the second sequence. For example, apolynucleotide comprising the wild-type sequence of a mutant gene ishomologous and non-identical to the sequence of the mutant gene. Incertain embodiments, the degree of homology between the two sequences issufficient to allow homologous recombination therebetween, utilizingnormal cellular mechanisms. Two homologous non-identical sequences canbe any length and their degree of non-homology can be as small as asingle nucleotide (e.g., for correction of a genomic point mutation bytargeted homologous recombination) or as large as 10 or more kilobases(e.g., for insertion of a gene at a predetermined ectopic site in achromosome). Two polynucleotides comprising the homologous non-identicalsequences need not be the same length. For example, an exogenouspolynucleotide (i.e., donor polynucleotide) of between 20 and 10,000nucleotides or nucleotide pairs can be used.

Techniques for determining nucleic acid and amino acid sequence identityare known in the art. Typically, such techniques include determining thenucleotide sequence of the mRNA for a gene and/or determining the aminoacid sequence encoded thereby, and comparing these sequences to a secondnucleotide or amino acid sequence. Genomic sequences can also bedetermined and compared in this fashion. In general, identity refers toan exact nucleotide-to-nucleotide or amino acid-to-amino acidcorrespondence of two polynucleotides or polypeptide sequences,respectively. Two or more sequences (polynucleotide or amino acid) canbe compared by determining their percent identity. The percent identityof two sequences, whether nucleic acid or amino acid sequences, is thenumber of exact matches between two aligned sequences divided by thelength of the shorter sequences and multiplied by 100. With respect tosequences described herein, the range of desired degrees of sequenceidentity is approximately 80% to 100% and any integer valuetherebetween. Typically the percent identities between sequences are atleast 70-75%, preferably 80-82%, more preferably 85-90%, even morepreferably 92%, still more preferably 95%, and most preferably 98%sequence identity.

Alternatively, the degree of sequence similarity between polynucleotidescan be determined by hybridization of polynucleotides under conditionsthat allow formation of stable duplexes between homologous regions,followed by digestion with single-stranded-specific nuclease(s), andsize determination of the digested fragments. Two nucleic acid, or twopolypeptide sequences are substantially homologous to each other whenthe sequences exhibit at least about 70%-75%, preferably 80%-82%, morepreferably 85%-90%, even more preferably 92%, still more preferably 95%,and most preferably 98% sequence identity over a defined length of themolecules, as determined using the methods above. As used herein,substantially homologous also refers to sequences showing completeidentity to a specified DNA or polypeptide sequence. DNA sequences thatare substantially homologous can be identified in a Southernhybridization experiment under, for example, stringent conditions, asdefined for that particular system. Defining appropriate hybridizationconditions is within the skill of the art. See, e.g., Sambrook et al.,supra; Nucleic Acid Hybridization: A Practical Approach, editors B. D.Hames and S. J. Higgins, (1985) Oxford; Washington, DC; IRL Press).

Selective hybridization of two nucleic acid fragments can be determinedas follows. The degree of sequence identity between two nucleic acidmolecules affects the efficiency and strength of hybridization eventsbetween such molecules. A partially identical nucleic acid sequence willat least partially inhibit the hybridization of a completely identicalsequence to a target molecule. Inhibition of hybridization of thecompletely identical sequence can be assessed using hybridization assaysthat are well known in the art (e.g., Southern (DNA) blot, Northern(RNA) blot, solution hybridization, or the like, see Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) ColdSpring Harbor, N.Y.). Such assays can be conducted using varying degreesof selectivity, for example, using conditions varying from low to highstringency. If conditions of low stringency are employed, the absence ofnon-specific binding can be assessed using a secondary probe that lackseven a partial degree of sequence identity (for example, a probe havingless than about 30% sequence identity with the target molecule), suchthat, in the absence of non-specific binding events, the secondary probewill not hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a reference nucleic acidsequence, and then by selection of appropriate conditions the probe andthe reference sequence selectively hybridize, or bind, to each other toform a duplex molecule. A nucleic acid molecule that is capable ofhybridizing selectively to a reference sequence under moderatelystringent hybridization conditions typically hybridizes under conditionsthat allow detection of a target nucleic acid sequence of at least about10-14 nucleotides in length having at least approximately 70% sequenceidentity with the sequence of the selected nucleic acid probe. Stringenthybridization conditions typically allow detection of target nucleicacid sequences of at least about 10-14 nucleotides in length having asequence identity of greater than about 90-95% with the sequence of theselected nucleic acid probe. Hybridization conditions useful forprobe/reference sequence hybridization, where the probe and referencesequence have a specific degree of sequence identity, can be determinedas is known in the art (see, for example, Nucleic Acid Hybridization: APractical Approach, editors B. D. Hames and S. J. Higgins, (1985)Oxford; Washington, DC; IRL Press).

Conditions for hybridization are well-known to those of skill in theart. Hybridization stringency refers to the degree to whichhybridization conditions disfavor the formation of hybrids containingmismatched nucleotides, with higher stringency correlated with a lowertolerance for mismatched hybrids. Factors that affect the stringency ofhybridization are well-known to those of skill in the art and include,but are not limited to, temperature, pH, ionic strength, andconcentration of organic solvents such as, for example, formamide anddimethylsulfoxide. As is known to those of skill in the art,hybridization stringency is increased by higher temperatures, lowerionic strength and lower solvent concentrations.

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of the sequences, base composition of thevarious sequences, concentrations of salts and other hybridizationsolution components, the presence or absence of blocking agents in thehybridization solutions (e.g., dextran sulfate, and polyethyleneglycol), hybridization reaction temperature and time parameters, as wellas, varying wash conditions. “Recombination” refers to a process ofexchange of genetic information between two polynucleotides. For thepurposes of this disclosure, “homologous recombination (HR)” refers tothe specialized form of such exchange that takes place, for example,during repair of double-strand breaks in cells. This process requiresnucleotide sequence homology, uses a “donor” molecule to template repairof a “target” molecule (i.e., the one that experienced the double-strandbreak), and is variously known as “non-crossover gene conversion” or“short tract gene conversion,” because it leads to the transfer ofgenetic information from the donor to the target. Without wishing to bebound by any particular theory, such transfer can involve mismatchcorrection of heteroduplex DNA that forms between the broken target andthe donor, and/or “synthesis-dependent strand annealing,” in which thedonor is used to resynthesize genetic information that will become partof the target, and/or related processes. Such specialized HR oftenresults in an alteration of the sequence of the target molecule suchthat part or all of the sequence of the donor polynucleotide isincorporated into the target polynucleotide.

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

An “accessible region” is a site in cellular chromatin in which a targetsite present in the nucleic acid can be bound by an exogenous moleculewhich recognizes the target site. Without wishing to be bound by anyparticular theory, it is believed that an accessible region is one thatis not packaged into a nucleosomal structure. The distinct structure ofan accessible region can often be detected by its sensitivity tochemical and enzymatic probes, for example, nucleases.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist. For example, thesequence 5′-GAATTC-3′ is a target site for the Eco RI restrictionendonuclease.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule, amalfunctioning version of a normally-functioning endogenous molecule oran ortholog (functioning version of endogenous molecule from a differentspecies).

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPDNA-binding domain and a cleavage domain) and fusion nucleic acids (forexample, a nucleic acid encoding the fusion protein described supra).Examples of the second type of fusion molecule include, but are notlimited to, a fusion between a triplex-forming nucleic acid and apolypeptide, and a fusion between a minor groove binder and a nucleicacid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of an mRNA. Gene products also include RNAswhich are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression. Gene inactivation refers to anyreduction in gene expression as compared to a cell that does not includea ZFP as described herein. Thus, gene inactivation may be partial orcomplete.

“Eukaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells(e.g., T-cells).

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a DNA-bindingdomain is fused to a cleavage domain, the DNA-binding domain and thecleavage domain are in operative linkage if, in the fusion polypeptide,the DNA-binding domain portion is able to bind its target site and/orits binding site, while the cleavage domain is able to cleave DNA in thevicinity of the target site (e.g., 1 to 500 base pairs or any valuetherebetween on either side of the target site).

In the methods of the disclosure, one or more targeted nucleases asdescribed herein create a double-stranded break (DSB) in the targetsequence (e.g., cellular chromatin) at a predetermined site. The DSB mayresult in deletions and/or insertions by homology-directed repair or bynon-homology-directed repair mechanisms. Deletions may include anynumber of base pairs. Similarly, insertions may include any number ofbase pairs including, for example, integration of a “donor”polynucleotide, optionally having homology to the nucleotide sequence inthe region of the break. The donor sequence may be physically integratedor, alternatively, the donor polynucleotide is used as a template forrepair of the break via homologous recombination, resulting in theintroduction of all or part of the nucleotide sequence as in the donorinto the cellular chromatin. Thus, a first sequence in cellularchromatin can be altered and, in certain embodiments, can be convertedinto a sequence present in a donor polynucleotide. Thus, the use of theterms “replace” or “replacement” can be understood to representreplacement of one nucleotide sequence by another, (i.e., replacement ofa sequence in the informational sense), and does not necessarily requirephysical or chemical replacement of one polynucleotide by another.

Additional pairs of zinc-finger proteins, TALENs, TtAgo or CRIPSR/Cassystems can be used for additional double-stranded cleavage ofadditional target sites within the cell.

In any of the methods described herein, additional pairs of zinc-fingerproteins, TALENs, TtAgo or CRIPSR/Cas systems can be used for additionaldouble-stranded cleavage of additional target sites within the cell.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain one ormore amino acid or nucleotide substitutions. Methods for determining thefunction of a nucleic acid (e.g., coding function, ability to hybridizeto another nucleic acid) are well-known in the art. Similarly, methodsfor determining protein function are well-known. For example, theDNA-binding function of a polypeptide can be determined, for example, byfilter-binding, electrophoretic mobility-shift, or immunoprecipitationassays. DNA cleavage can be assayed by gel electrophoresis. See Ausubelet al., supra. The ability of a protein to interact with another proteincan be determined, for example, by co-immunoprecipitation, two-hybridassays or complementation, both genetic and biochemical. See, forexample, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No.5,585,245 and PCT WO 98/44350.

Linkers

Described herein are amino acid sequences that fuse (link) a DNA bindingdomain (e.g., zinc finger protein, TALE, etc.) and a nuclease (e.g., acleavage domain or cleavage half-domain).

Currently, when a pair of nucleases is used to cleave a genomicsequence, optimal cleavage is obtained when the DNA-binding proteinsbind to target sites separated by less than 6 base pairs. In particular,optimal cleavage for zinc finger nuclease pairs is obtained when thetarget sites are separated by 5-6 base pairs and a flexible “ZC” linkerrich in glycine and serine is used to join each zinc finger of the pairto the cleavage domain. In particular, the “ZC” linker used to dateconsists of the amino acid sequence LRGS (SEQ ID NO:35) between theC-terminal of the zinc finger binding domain and the N-terminal residuesof the cleavage domain, which in the case of FokI is a Q residue. See,e.g., U.S. Pat. No. 7,888,121. In addition, fusion proteins comprisingZC linkers and additional modifications to the N-terminal region of thecleavage domain have also been described. See, U.S. Patent PublicationNo. 20090305419. Furthermore, U.S. Pat. No. 8,772,453 describes linkersuseful for obtaining cleavage when the DNA-binding sites targetsequences separated by less than 5 base pairs.

The linkers described herein allow cleavage when the target sites of apair of zinc finger nucleases are not 0-6 base pairs apart, for exampletarget sites that are 7, 8, 9, 10 or more base pairs apart. The linkersequences described herein are typically between about 8 and 17 aminoacids in length and may link the N- or C-terminal of the DNA-bindingdomain to the N- or C-terminal of cleavage domain. In certainembodiments, the linker extends between the C-terminal residue of theDNA-binding domain and the N-terminal residue of the cleavage domain(e.g., (Q) of FokI.

Non-limiting examples of linkers as described herein are shown in FIGS.4, 7, 9, 11, 12, 13, 15 and 16 (SEQ ID NOs: 2-21, 45-49, 51, 62-226,233-240, and 258-312) (with or without 1 or 2 of the C-terminal aminoacid residues shown in these Figures), including but not limited to(N)GICPPPRPRTSPP (SEQ ID NO:2); (T)GTAPIEIPPEVYP (SEQ ID NO:3);(N)GSYAPMPPLALASP (SEQ ID NO:4); (P)GIYTAPTSRPTVPP (SEQ ID NO:5);(N)GSQTPKRFQPTHPSA (SEQ ID NO:6); (H)LPKPANPFPLD (SEQ ID NO:7);(H)RDGPRNLPPTSPP (SEQ ID NO:8); (H)RLPDSPTALAPDTL (SEQ ID NO:9);(DPNS)PISRARPLNPHP (SEQ ID NO:10); (Y)GPRPTPRLRCPIDSLIFR (SEQ ID NO:11);(H)CPASRPIHP (SEQ ID NO:12); (G)LQSLIPQQLL (SEQ ID NO:13);(G)LQPTVNHEYNN (SEQ ID NO:14); (P)ANIHSLSSPPPL (SEQ ID NO:15);(P)AGLNTPCSPRSRSN (SEQ ID NO:16); (A)TITDPNP (SEQ ID NO:17); (P)PHKGLLP(SEQ ID NO:18); (S)VSLPDTHH(SEQ ID NO:19); (G)THGATPTHSP (SEQ ID NO:20);(V)APGESSMTSL (SEQ ID NO:21), (LRGS)PISRARPLNPHP (SEQ ID NO:22),(LRGS)ISRARPLNPHP (SEQ ID NO:23), (LRGS)PSRARPLNPHP (SEQ ID NO:24),(LRGS)SRARPLNPHP (SEQ ID NO:25), (LRGS)YAPMPPLALASP (SEQ ID NO:26),(LRGS)APMPPLALASP (SEQ ID NO:27), (LRGS)PMPPLALASP (SEQ ID NO:28),(LRGS)MPPLALASP (SEQ ID NO:29) HLPKPANPFPLD (SEQ ID NO:30), wherein theamino acid residue(s) shown in round parentheses is(are) optionallypresent. In certain embodiments, the linker comprises a sequenceselected from the group consisting of PKPAN (SEQ ID NO:31), RARPLN (SEQID NO:32); PMPPLA (SEQ ID NO:33) or PPPRP (SEQ ID NO:34). In certainembodiments, the linkers further comprise a ZC linker (LRGS, SEQ IDNO:35) at their N-terminal.

In certain embodiments, the linker comprises a sequence as shown in FIG.4 (SEQ ID NOs:62-226, respectively, in order of appearance). In otherembodiments, the linker comprises a sequence as shown in FIG. 7, FIG. 9A(top), FIG. 12 and FIG. 13 (L8 linkers including, for example, L8a (SEQID NO: 2), L8b (SEQ ID NO: 3), L8c (SEQ ID NO: 4), L8d (SEQ ID NO: 5),L8e (SEQ ID NO: 6), L8g (SEQ ID NO: 234), L8i (SEQ ID NO: 161), L8j (SEQID NO: 235), L8a (SEQ ID NO: 2), L8c (SEQ ID NO: 4), L8a3 (SEQ ID NO:268) and L8c3 (SEQ ID NO: 269),). In certain embodiments, the L8 linkersare used when the DNA-binding domains of the dimerizing nuclease pairused for cleavage bind to target sites separate by 8 base pairs. Inother embodiments, the linker comprises a sequence as shown in FIG. 9A(bottom), FIG. 12 and FIG. 13 (L7 linkers including, for example, L7-1(SEQ ID NO: 7), L7-2 (SEQ ID NO: 8), L7-6 (SEQ ID NO: 9), L7-3 (SEQ IDNO: 10), L7-5 (SEQ ID NO: 11), L7-4 (SEQ ID NO: 236), L7-b (SEQ ID NO:258), L7-c (SEQ ID NO: 259), L7-b3 (SEQ ID NO: 266) and L7-c3 (SEQ IDNO: 267),). In certain embodiments, the L7 linkers are used when theDNA-binding domains of the dimerizing nuclease pair used for cleavagebind to target sites separate by 7 base pairs. In other embodiments, thelinker comprises a sequence as shown in FIG. 9B (top) (L6 linkers,including, for example, L6-2 (SEQ ID NO: 12), L6-6 (SEQ ID NO: 13), L6-7(SEQ ID NO: 14), L6-1 (SEQ ID NO: 15), L6-5 (SEQ ID NO: 16), L6-3 (SEQID NO: 115), or L6-4 (SEQ ID NO: 237),). In certain embodiments, the L6linkers are used when the DNA-binding domains of the dimerizing nucleasepair used for cleavage bind to target sites separate by 6 base pairs. Instill other embodiments, the linker comprises a sequence as shown inFIG. 9B (bottom) (L5-6 (SEQ ID NO: 17), L5-1 (SEQ ID NO: 18), L5-7 (SEQID NO: 19), L5-8 (SEQ ID NO: 20), L5-9 (SEQ ID NO: 21), L5-2 (SEQ ID NO:238), L5-3 (SEQ ID NO: 239), L5-4 (SEQ ID NO: 240), L5-5 (SEQ ID NO:71),). In certain embodiments, the L5 linkers are used when theDNA-binding domains of the dimerizing nuclease pair used for cleavagebind to target sites separate by 5 base pairs.

The fusion proteins described herein may also include alterations to theN-terminal region of the selected cleavage domain. Alteration mayinclude substitutions, additions and/or deletions of one or moreN-terminal residues of the cleavage domain. In certain embodiments, thecleavage domain is derived from FokI and one or more amino acids of thewild-type FokI N-terminal region are replaced and additional amino acidsadded to this region. For example, as shown in FIG. 2, amino acidresidues 4 and 5 of the wild-type FokI cleavage domain (i.e., residues Kand S) are replaced with residues E and A, respectively and the residuesAAR is added C-terminal to the 2^(nd) replaced residue. Anotherexemplary embodiment (FIG. 3) includes a seven residue insertion(KSEAAAR; SEQ ID NO:36) in the N-terminal region of the FokI cleavagedomain.

The sequence joining the DNA-binding domain and the cleavage domain cancomprise any amino acid sequence that does not substantially hinder theability of the DNA-binding domain to bind to its target site or thecleavage domain to dimerize and/or cleave the genomic sequences. Inwild-type FokI, the N-terminal region of the cleavage domain includes analpha helical region extending from residues 389-400 (ELEEKKSELRHK; SEQID NO:37). See, e.g., Wah et al. (1997) Nature 388:97-100). Therefore,in certain embodiments, the linker sequences are designed to extendand/or conserve this structural motif, for example by inserting a 3-5amino sequence N-terminal to ELEEKKSELRHK (SEQ ID NO:37) of a wild-typeFokI cleavage domain.

Thus, the linker may include a sequence such as EXXXR (SEQ ID NO:38) orEXXXK (SEQ ID NO:39) where the X residues are any residues that form analpha helix, namely any residue except proline or glycine (e.g., EAAAR(SEQ ID NO:40)) adjacent to the wild-type alpha helical region to form astable alpha helix linker. See, e.g., Yan et al. (2007) Biochemistry46:8517-24 and Merutka and Stellwagen (1991) Biochemistry 30:4245-8.Placing an EXXXR (SEQ ID NO:38) or EXXXK (SEQ ID NO:39) peptide adjacent(or near to) to the ELEEKKSELRHK (SEQ ID NO:37) peptide is designed toextend this alpha helix in FokI cleavage domain. This creates a morerigid linker between the ZFP and FokI cleavage domain which allows theresulting ZFN pair to cleave a target with more than 6 bp between thehalf sites without the loss in activity and specificity that can beobserved when a long flexible linker is used between the ZFP and theFokI domain (Bibikova et al. (2001) Molecular and Cellular Biology21:289-297). In addition, the linkers described herein show a greaterpreference for a 6 bp spacing over a 5 bp spacing as compared to currentZFNs.

Furthermore, in certain embodiments, the junction as between the linkerand the cleavage domain and/or DNA-binding domain is modified, forexample to substitute, add and/or delete amino acids to the linkers asdescribed herein. In certain embodiments, 1, 2, 3, 4 or more amino acidsare deleted or added. In other embodiment, 1, 2 or 3, 4 amino acidsubstitutions are made. Non-limiting examples of modified linkers asdescribed herein are shown in FIGS. 15 and 16 (SEQ ID NOs:280-312 and51).

Also described herein is a fusion protein comprising a DNA-bindingdomain, a modified FokI cleavage domain and a ZC linker between theDNA-binding domain and the FokI cleavage domain. The FokI cleavagedomain may be modified in any way. Non-limiting examples ofmodifications include additions, deletions and/or substitutions to theN-terminal region of FokI (residues 158-169 of SEQ ID NO:1). In certainembodiments, the modified FokI cleavage domain comprises deletion of 1,2, 3, 4 or more amino acids from the N-terminal region of FokI (e.g.,deletion of one or more of residues 158, 159, 160 and/or 161 of thewild-type FokI domain of SEQ ID NO:1). Non-limiting examples of proteinswith deletions of N-terminal FokI amino acid residues include theproteins designated V1, V3 and V7 as shown in FIG. 15. In otherembodiments, the modified FokI cleavage domain comprises one or moredeletions and one or more substitutions from the N-terminal region ofFokI (e.g., deletion of one or more of residues 158-161 and substitutionof one or more of the remaining residues). Non-limiting examples ofproteins with deletions and substitutions in the N-terminal FokI aminoacid residues include the proteins designated V2, V4, V5, V6 and V8 asshown in FIG. 15. In other embodiments, the modified FokI cleavagedomain comprises one or more substitutions in the N-terminal region ofFokI. Non-limiting examples of proteins with substitutions in theN-terminal amino acid residues of FokI include the proteins designatedV9 through V16 as shown in FIG. 15. In still further embodiments, themodified FokI cleavage domain comprises one or more additional aminoacid residues (e.g., 1, 2, 3, 4 or more)N-terminal to theN-terminal-most residue of FokI (residue 158 of SEQ ID NO:1).Non-limiting examples of proteins with additions to the N-terminal FokIamino acid residues include the proteins designated V17 through V24 asshown in FIG. 15. In other embodiments, the modified FokI cleavagedomain comprises one or more additional amino acid residues (e.g., 1, 2,3, 4 or more)N-terminal to the N-terminal-most residue of FokI (residue158 of SEQ ID NO:1) and one or more substitutions within the N-terminalregion of FokI. Non-limiting examples of proteins with additions includethose shown in FIG. 15 or FIG. 16 and substitutions within theN-terminal FokI amino acid residues as described in U.S. PatentPublication No 20090305419. In certain embodiments, the fusion proteincomprises a modified FokI cleavage domain as shown in FIG. 15 or FIG.16.

Typically, the linkers of the invention are made by making recombinantnucleic acids encoding the linker and the DNA-binding domains, which arefused via the linker amino acid sequence. Optionally, the linkers canalso be made using peptide synthesis, and then linked to the polypeptideDNA-binding domains.

Nucleases

The linker sequences described herein are advantageously used to linkDNA-binding domains, for example zinc finger proteins, TALEs, homingendonucleases, CRISPR/Cas and/or Ttago guide RNAs, to nuclease cleavagedomains or half domains to form specifically targeted, non-naturallyoccurring nucleases.

A. DNA-Binding Domains

Any DNA-binding domain can be used in the methods disclosed herein. Incertain embodiments, the DNA binding domain comprises a zinc fingerprotein. Preferably, the zinc finger protein is non-naturally occurringin that it is engineered to bind to a target site of choice. See, forexample, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al.(2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) NatureBiotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol.12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416. Anengineered zinc finger binding domain can have a novel bindingspecificity, compared to a naturally-occurring zinc finger protein.Engineering methods include, but are not limited to, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242and 6,534,261, incorporated by reference herein in their entireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

Selection of target sites; ZFPs and methods for design and constructionof fusion proteins (and polynucleotides encoding same) are known tothose of skill in the art and described in detail in U.S. PatentPublication Nos. 20050064474 and 20060188987, incorporated by referencein their entireties herein.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

In certain embodiments, the composition and methods described hereinemploy a meganuclease (homing endonuclease) DNA-binding domain forbinding to the donor molecule and/or binding to the region of interestin the genome of the cell. Naturally-occurring meganucleases recognize15-40 base-pair cleavage sites and are commonly grouped into fourfamilies: the LAGLIDADG family (‘LAGLIDADG’ disclosed as SEQ ID NO: 41),the GIY-YIG family, the His-Cyst box family and the HNH family.Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce,I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI,I-TevII and I-TevIII. Their recognition sequences are known. See alsoU.S. Pat. Nos. 5,420,032; 6,833,252; Belfort et al. (1997) Nucleic AcidsRes. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al.(1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet.12:224-228; Gimble et al. (1996) J. Mol. Biol. 263:163-180; Argast etal. (1998) J. Mol. Biol. 280:345-353 and the New England Biolabscatalogue. In addition, the DNA-binding specificity of homingendonucleases and meganucleases can be engineered to bind non-naturaltarget sites. See, for example, Chevalier et al. (2002) Molec. Cell10:895-905; Epinat et al. (2003) Nucleic Acids Res. 31:2952-2962;Ashworth et al. (2006) Nature 441:656-659; Paques et al. (2007) CurrentGene Therapy 7:49-66; U.S. Patent Publication No. 20070117128. TheDNA-binding domains of the homing endonucleases and meganucleases may bealtered in the context of the nuclease as a whole (i.e., such that thenuclease includes the cognate cleavage domain) or may be fused to aheterologous cleavage domain.

In other embodiments, the DNA-binding domain of one or more of thenucleases used in the methods and compositions described hereincomprises a naturally occurring or engineered (non-naturally occurring)TAL effector DNA binding domain. See, e.g., U.S. Pat. No. 8,586,526,incorporated by reference in its entirety herein. The plant pathogenicbacteria of the genus Xanthomonas are known to cause many diseases inimportant crop plants. Pathogenicity of Xanthomonas depends on aconserved type III secretion (T3 S) system which injects more than 25different effector proteins into the plant cell. Among these injectedproteins are transcription activator-like (TAL) effectors which mimicplant transcriptional activators and manipulate the plant transcriptome(see Kay et al (2007) Science 318:648-651). These proteins contain a DNAbinding domain and a transcriptional activation domain. One of the mostwell characterized TAL-effectors is AvrBs3 from Xanthomonas campestgrispv. Vesicatoria (see Bonas et al (1989) Mol Gen Genet 218: 127-136 andWO2010079430). TAL-effectors contain a centralized domain of tandemrepeats, each repeat containing approximately 34 amino acids, which arekey to the DNA binding specificity of these proteins. In addition, theycontain a nuclear localization sequence and an acidic transcriptionalactivation domain (for a review see Schornack S, et al (2006) J PlantPhysiol 163(3): 256-272). In addition, in the phytopathogenic bacteriaRalstonia solanacearum two genes, designated brgll and hpx17 have beenfound that are homologous to the AvrBs3 family of Xanthomonas in the R.solanacearum biovar 1 strain GMI1000 and in the biovar 4 strain RS1000(See Heuer et al (2007) Appl and Envir Micro 73(13): 4379-4384). Thesegenes are 98.9% identical in nucleotide sequence to each other butdiffer by a deletion of 1,575 bp in the repeat domain of hpx17. However,both gene products have less than 40% sequence identity with AvrBs3family proteins of Xanthomonas. See, e.g., U.S. Pat. No. 8,586,526,incorporated by reference in its entirety herein.

Specificity of these TAL effectors depends on the sequences found in thetandem repeats. The repeated sequence comprises approximately 102 bp andthe repeats are typically 91-100% homologous with each other (Bonas etal, ibid). Polymorphism of the repeats is usually located at positions12 and 13 and there appears to be a one-to-one correspondence betweenthe identity of the hypervariable diresidues (RVD) at positions 12 and13 with the identity of the contiguous nucleotides in the TAL-effector'starget sequence (see Moscou and Bogdanove, (2009) Science 326:1501 andBoch et al (2009) Science 326:1509-1512). Experimentally, the naturalcode for DNA recognition of these TAL-effectors has been determined suchthat an HD sequence at positions 12 and 13 leads to a binding tocytosine (C), NG binds to T, NI to A, C, G or T, NN binds to A or G, andING binds to T. These DNA binding repeats have been assembled intoproteins with new combinations and numbers of repeats, to makeartificial transcription factors that are able to interact with newsequences and activate the expression of a non-endogenous reporter genein plant cells (Boch et al, ibid). Engineered TAL proteins have beenlinked to a FokI cleavage half domain to yield a TAL effector domainnuclease fusion (TALEN). See, e.g., U.S. Pat. No. 8,586,526; Christianet al ((2010)<Geneticsepub 10.1534/genetics.110.120717). In certainembodiments, TALE domain comprises an N-cap and/or C-cap as described inU.S. Pat. No. 8,586,526. In still further embodiments, the nucleasecomprises a compact TALEN (cTALEN). These are single chain fusionproteins linking a TALE DNA binding domain to a TevI nuclease domain.The fusion protein can act as either a nickase localized by the TALEregion, or can create a double strand break, depending upon where theTALE DNA binding domain is located with respect to the TevI nucleasedomain (see Beurdeley et al (2013) Nat Comm: 1-8 DOI:10.1038/ncomms2782). Any TALENs may be used in combination withadditional TALENs (e.g., one or more TALENs (cTALENs or FokI-TALENs)with one or more mega-TALs).

In certain embodiments, the DNA-binding domain is part of a CRISPR/Casnuclease system. See, e.g., U.S. Pat. No. 8,697,359 and U.S. PatentPublication No. 20150056705. The CRISPR (clustered regularly interspacedshort palindromic repeats) locus, which encodes RNA components of thesystem, and the cas (CRISPR-associated) locus, which encodes proteins(Jansen et al., 2002. Mol. Microbiol. 43: 1565-1575; Makarova et al.,2002. Nucleic Acids Res. 30: 482-496; Makarova et al., 2006. Biol.Direct 1: 7; Haft et al., 2005. PLoSComput. Biol. 1: e60) make up thegene sequences of the CRISPR/Cas nuclease system. CRISPR loci inmicrobial hosts contain a combination of CRISPR-associated (Cas) genesas well as non-coding RNA elements capable of programming thespecificity of the CRISPR-mediated nucleic acid cleavage.

The Type II CRISPR is one of the most well characterized systems andcarries out targeted DNA double-strand break in four sequential steps.First, two non-coding RNA, the pre-crRNA array and tracrRNA, aretranscribed from the CRISPR locus. Second, tracrRNA hybridizes to therepeat regions of the pre-crRNA and mediates the processing of pre-crRNAinto mature crRNAs containing individual spacer sequences. Third, themature crRNA:tracrRNA complex directs Cas9 to the target DNA viaWatson-Crick base-pairing between the spacer on the crRNA and theprotospacer on the target DNA next to the protospacer adjacent motif(PAM), an additional requirement for target recognition. Finally, Cas9mediates cleavage of target DNA to create a double-stranded break withinthe protospacer. Activity of the CRISPR/Cas system comprises of threesteps: (i) insertion of alien DNA sequences into the CRISPR array toprevent future attacks, in a process called ‘adaptation’, (ii)expression of the relevant proteins, as well as expression andprocessing of the array, followed by (iii) RNA-mediated interferencewith the alien nucleic acid. Thus, in the bacterial cell, several of theso-called ‘Cas’ proteins are involved with the natural function of theCRISPR/Cas system and serve roles in functions such as insertion of thealien DNA etc.

In certain embodiments, Cas protein may be a “functional derivative” ofa naturally occurring Cas protein. A “functional derivative” of a nativesequence polypeptide is a compound having a qualitative biologicalproperty in common with a native sequence polypeptide. “Functionalderivatives” include, but are not limited to, fragments of a nativesequence and derivatives of a native sequence polypeptide and itsfragments, provided that they have a biological activity in common witha corresponding native sequence polypeptide. A biological activitycontemplated herein is the ability of the functional derivative tohydrolyze a DNA substrate into fragments. The term “derivative”encompasses both amino acid sequence variants of polypeptide, covalentmodifications, and fusions thereof. Suitable derivatives of a Caspolypeptide or a fragment thereof include but are not limited tomutants, fusions, covalent modifications of Cas protein or a fragmentthereof. Cas protein, which includes Cas protein or a fragment thereof,as well as derivatives of Cas protein or a fragment thereof, may beobtainable from a cell or synthesized chemically or by a combination ofthese two procedures. The cell may be a cell that naturally produces Casprotein, or a cell that naturally produces Cas protein and isgenetically engineered to produce the endogenous Cas protein at a higherexpression level or to produce a Cas protein from an exogenouslyintroduced nucleic acid, which nucleic acid encodes a Cas that is sameor different from the endogenous Cas. In some case, the cell does notnaturally produce Cas protein and is genetically engineered to produce aCas protein.

In some embodiments, the DNA binding domain is part of a TtAgo system(see Swarts et al, ibid; Sheng et al, ibid). In eukaryotes, genesilencing is mediated by the Argonaute (Ago) family of proteins. In thisparadigm, Ago is bound to small (19-31 nt) RNAs. This protein-RNAsilencing complex recognizes target RNAs via Watson-Crick base pairingbetween the small RNA and the target and endonucleolytically cleaves thetarget RNA (Vogel (2014) Science 344:972-973). In contrast, prokaryoticAgo proteins bind to small single-stranded DNA fragments and likelyfunction to detect and remove foreign (often viral) DNA (Yuan et al.,(2005) Mol. Cell 19, 405; Olovnikov, et al. (2013) Mol. Cell 51, 594;Swarts et al., ibid). Exemplary prokaryotic Ago proteins include thosefrom Aquifex aeolicus, Rhodobacter sphaeroides, and Thermusthermophilus.

One of the most well-characterized prokaryotic Ago protein is the onefrom T. thermophilus (TtAgo; Swarts et al. ibid). TtAgo associates witheither 15 nt or 13-25 nt single-stranded DNA fragments with 5′ phosphategroups. This “guide DNA” bound by TtAgo serves to direct the protein-DNAcomplex to bind a Watson-Crick complementary DNA sequence in athird-party molecule of DNA. Once the sequence information in theseguide DNAs has allowed identification of the target DNA, the TtAgo-guideDNA complex cleaves the target DNA. Such a mechanism is also supportedby the structure of the TtAgo-guide DNA complex while bound to itstarget DNA (G. Sheng et al., ibid). Ago from Rhodobacter sphaeroides(RsAgo) has similar properties (Olivnikov et al. ibid).

Exogenous guide DNAs of arbitrary DNA sequence can be loaded onto theTtAgo protein (Swarts et al. ibid.). Since the specificity of TtAgocleavage is directed by the guide DNA, a TtAgo-DNA complex formed withan exogenous, investigator-specified guide DNA will therefore directTtAgo target DNA cleavage to a complementary investigator-specifiedtarget DNA. In this way, one may create a targeted double-strand breakin DNA. Use of the TtAgo-guide DNA system (or orthologous Ago-guide DNAsystems from other organisms) allows for targeted cleavage of genomicDNA within cells. Such cleavage can be either single- ordouble-stranded. For cleavage of mammalian genomic DNA, it would bepreferable to use of a version of TtAgo codon optimized for expressionin mammalian cells. Further, it might be preferable to treat cells witha TtAgo-DNA complex formed in vitro where the TtAgo protein is fused toa cell-penetrating peptide. Further, it might be preferable to use aversion of the TtAgo protein that has been altered via mutagenesis tohave improved activity at 37° C. Ago-RNA-mediated DNA cleavage could beused to effect a panopoly of outcomes including gene knock-out, targetedgene addition, gene correction, targeted gene deletion using techniquesstandard in the art for exploitation of DNA breaks.

B. Cleavage Domains

The nucleases described herein (e.g., ZFNs, TALENs, CRISPR/Cas nuclease)also comprise a nuclease (cleavage domain, cleavage half-domain). Thecleavage domain portion of the fusion proteins disclosed herein can beobtained from any endonuclease or exonuclease. Exemplary endonucleasesfrom which a cleavage domain can be derived include, but are not limitedto, restriction endonucleases and homing endonucleases. See, forexample, 2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; andBelfort et al. (1997) Nucleic Acids Res. 25:3379-3388. Additionalenzymes which cleave DNA are known (e.g., Si Nuclease; mung beannuclease; pancreatic DNase I; micrococcal nuclease; yeast HOendonuclease; see also Linn et al. (eds.) Nucleases, Cold Spring HarborLaboratory Press, 1993). One or more of these enzymes (or functionalfragments thereof) can be used as a source of cleavage domains andcleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof).

In addition, the target sites for the two fusion proteins are preferablydisposed, with respect to each other, such that binding of the twofusion proteins to their respective target sites places the cleavagehalf-domains in a spatial orientation to each other that allows thecleavage half-domains to form a functional cleavage domain, e.g., bydimerizing. Thus, in certain embodiments, the near edges of the targetsites are separated by 5-8 nucleotides or by 15-18 nucleotides. Howeverany integral number of nucleotides or nucleotide pairs can intervenebetween two target sites (e.g., from 2 to 50 nucleotide pairs or more).In general, the site of cleavage lies between the target sites.

As noted above, the cleavage domain may be heterologous to theDNA-binding domain, for example a zinc finger DNA-binding domain and acleavage domain from a nuclease or a TALEN DNA-binding domain and acleavage domain, or meganuclease DNA-binding domain and cleavage domainfrom a different nuclease, or a DNA binding domain from a CRISPR/Cassystem and a cleavage domain from a difference nuclease. Heterologouscleavage domains can be obtained from any endonuclease or exonuclease.Exemplary endonucleases from which a cleavage domain can be derivedinclude, but are not limited to, restriction endonucleases and homingendonucleases. Additional enzymes which cleave DNA are known (e.g., SiNuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease;yeast HO endonuclease. One or more of these enzymes (or functionalfragments thereof) can be used as a source of cleavage domains andcleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral number of nucleotides or nucleotidepairs can intervene between two target sites (e.g., from 2 to 50nucleotide pairs or more). In general, the site of cleavage lies betweenthe target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme FokI catalyzes double-strandedcleavage of DNA, at 9 nucleotides from its recognition site on onestrand and 13 nucleotides from its recognition site on the other. See,for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as wellas Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al.(1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc.Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem.269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise thecleavage domain (or cleavage half-domain) from at least one Type IISrestriction enzyme and one or more zinc finger binding domains, whichmay or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is FokI. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the FokI enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-FokI fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a zinc finger binding domain and twoFokI cleavage half-domains can also be used. Parameters for targetedcleavage and targeted sequence alteration using zinc finger-FokI fusionsare provided elsewhere in this disclosure.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPublication WO 07/014275, incorporated herein in its entirety.Additional restriction enzymes also contain separable binding andcleavage domains, and these are contemplated by the present disclosure.See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Pat. Nos. 8,623,618; 8,409,861; 8,034,598; 7,914,796;and 7,888,121, the disclosures of all of which are incorporated byreference in their entireties herein. Amino acid residues at positions446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531,534, 537, and 538 of Fok I are all targets for influencing dimerizationof the Fok I cleavage half-domains.

Exemplary engineered cleavage half-domains of Fok I that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFok I and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499.

Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys(K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:I538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:I499L”. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished. See, e.g., U.S.Pat. No. 7,888,121, the disclosure of which is incorporated by referencein its entirety for all purposes.

Cleavage domains with more than one mutation may be used, for examplemutations at positions 490 (E→K) and 538 (I→K) in one cleavagehalf-domain to produce an engineered cleavage half-domain designated“E490K:I538K” and by mutating positions 486 (Q→E) and 499 (I→L) inanother cleavage half-domain to produce an engineered cleavagehalf-domain designated “Q486E:I499L;” mutations that replace the wildtype Gln (Q) residue at position 486 with a Glu (E) residue, the wildtype Iso (I) residue at position 499 with a Leu (L) residue and thewild-type Asn (N) residue at position 496 with an Asp (D) or Glu (E)residue (also referred to as a “ELD” and “ELE” domains, respectively);engineered cleavage half-domain comprising mutations at positions 490,538 and 537 (numbered relative to wild-type FokI), for instancemutations that replace the wild type Glu (E) residue at position 490with a Lys (K) residue, the wild type Iso (I) residue at position 538with a Lys (K) residue, and the wild-type His (H) residue at position537 with a Lys (K) residue or a Arg (R) residue (also referred to as“KKK” and “KKR” domains, respectively); and/or engineered cleavagehalf-domain comprises mutations at positions 490 and 537 (numberedrelative to wild-type FokI), for instance mutations that replace thewild type Glu (E) residue at position 490 with a Lys (K) residue and thewild-type His (H) residue at position 537 with a Lys (K) residue or aArg (R) residue (also referred to as “KIK” and “KIR” domains,respectively). See, e.g., U.S. Pat. Nos. 7,914,796; 8,034,598 and8,623,618, the disclosures of which are incorporated by reference in itsentirety for all purposes. In other embodiments, the engineered cleavagehalf domain comprises the “Sharkey” and/or “Sharkey” mutations (see Guoet al, (2010) J. Mol. Biol. 400(1):96-107).

Alternatively, nucleases may be assembled in vivo at the nucleic acidtarget site using so-called “split-enzyme” technology (see e.g. U.S.Patent Publication No. 20090068164). Components of such split enzymesmay be expressed either on separate expression constructs, or can belinked in one open reading frame where the individual components areseparated, for example, by a self-cleaving 2A peptide or IRES sequence.Components may be individual zinc finger binding domains or domains of ameganuclease nucleic acid binding domain.

Nucleases can be screened for activity prior to use, for example in ayeast-based chromosomal system as described in U.S. Pat. No. 8,563,314.

The Cas9 related CRISPR/Cas system comprises two RNA non-codingcomponents: tracrRNA and a pre-crRNA array containing nuclease guidesequences (spacers) interspaced by identical direct repeats (DRs). Touse a CRISPR/Cas system to accomplish genome engineering, both functionsof these RNAs must be present (see Cong et al, (2013) Sciencexpress1/10.1126/science 1231143). In some embodiments, the tracrRNA andpre-crRNAs are supplied via separate expression constructs or asseparate RNAs. In other embodiments, a chimeric RNA is constructed wherean engineered mature crRNA (conferring target specificity) is fused to atracrRNA (supplying interaction with the Cas9) to create a chimericcr-RNA-tracrRNA hybrid (also termed a single guide RNA). (see Jinek ibidand Cong, ibid).

Target Sites

As described in detail above, DNA-binding domains of the fusion proteinscomprising the linkers as described herein can be engineered to bind toany sequence of choice. An engineered DNA-binding domain can have anovel binding specificity, compared to a naturally-occurring DNA-bindingdomain.

Non-limiting examples of suitable target genes a beta (β) globin gene(HBB), a gamma (δ) globin gene (HBG1), a B-cell lymphoma/leukemia 11A(BCL11A) gene, a Kruppel-like factor 1 (KLF1) gene, a CCR5 gene, a CXCR4gene, a PPP1R12C (AAVS1) gene, an hypoxanthine phosphoribosyltransferase(HPRT) gene, an albumin gene, a Factor VIII gene, a Factor IX gene, aLeucine-rich repeat kinase 2 (LRRK2) gene, a Hungtingin (Htt) gene, arhodopsin (RHO) gene, a Cystic Fibrosis Transmembrane ConductanceRegulator (CFTR) gene, a surfactant protein B gene (SFTPB), a T-cellreceptor alpha (TRAC) gene, a T-cell receptor beta (TRBC) gene, aprogrammed cell death 1 (PD1) gene, a Cytotoxic T-Lymphocyte Antigen 4(CTLA-4) gene, an human leukocyte antigen (HLA) A gene, an HLA B gene,an HLA C gene, an HLA-DPA gene, an HLA-DQ gene, an HLA-DRA gene, a LMP7gene, a Transporter associated with Antigen Processing (TAP) 1 gene, aTAP2 gene, a tapasin gene (TAPBP), a class II major histocompatibilitycomplex transactivator (CIITA) gene, a dystrophin gene (DMD), aglucocorticoid receptor gene (GR), an IL2RG gene, a Rag-1 gene, an RFX5gene, a FAD2 gene, a FAD3 gene, a ZP15 gene, a KASII gene, a MDH gene,and/or an EPSPS gene.

In certain embodiments, the nuclease targets a “safe harbor” loci suchas the AAVS1, HPRT, albumin and CCR5 genes in human cells, and Rosa26 inmurine cells (see, e.g., U.S. Pat. Nos. 7,888,121; 7,972,854; 7,914,796;7,951,925; 8,110,379; 8,409,861; 8,586,526; U.S. Patent Publications20030232410; 20050208489; 20050026157; 20060063231; 20080159996;201000218264; 20120017290; 20110265198; 20130137104; 20130122591;20130177983 and 20130177960) and the Zp15 locus in plants (see U.S. Pat.No. 8,329,986).

Donors

In certain embodiments, the present disclosure relates tonuclease-mediated modification of the genome of a stem cell. As notedabove, insertion of an exogenous sequence (also called a “donorsequence” or “donor” or “transgene”), for example for deletion of aspecified region and/or correction of a mutant gene or for increasedexpression of a wild-type gene. It will be readily apparent that thedonor sequence is typically not identical to the genomic sequence whereit is placed. A donor sequence can contain a non-homologous sequenceflanked by two regions of homology to allow for efficient HDR at thelocation of interest or can be integrated via non-homology directedrepair mechanisms. Additionally, donor sequences can comprise a vectormolecule containing sequences that are not homologous to the region ofinterest in cellular chromatin. A donor molecule can contain several,discontinuous regions of homology to cellular chromatin. Further, fortargeted insertion of sequences not normally present in a region ofinterest, said sequences can be present in a donor nucleic acid moleculeand flanked by regions of homology to sequence in the region ofinterest.

As with nucleases, the donors can be introduced in any form. In certainembodiments, the donors are introduced in mRNA form to eliminateresidual virus in the modified cells. In other embodiments, the donorsmay be introduced using DNA and/or viral vectors by methods known in theart. See, e.g., U.S. Patent Publication Nos. 20100047805 and20110207221. The donor may be introduced into the cell in circular orlinear form. If introduced in linear form, the ends of the donorsequence can be protected (e.g., from exonucleolytic degradation) bymethods known to those of skill in the art. For example, one or moredideoxynucleotide residues are added to the 3′ terminus of a linearmolecule and/or self-complementary oligonucleotides are ligated to oneor both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad.Sci. USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889.Additional methods for protecting exogenous polynucleotides fromdegradation include, but are not limited to, addition of terminal aminogroup(s) and the use of modified internucleotide linkages such as, forexample, phosphorothioates, phosphoramidates, and O-methyl ribose ordeoxyribose residues.

In certain embodiments, the donor includes sequences (e.g., codingsequences, also referred to as transgenes) greater than 1 kb in length,for example between 2 and 200 kb, between 2 and 10 kb (or any valuetherebetween). The donor may also include at least one nuclease targetsite. In certain embodiments, the donor includes at least 2 targetsites, for example for a pair of ZFNs, TALENs, TtAgo or CRISPR/Casnucleases. Typically, the nuclease target sites are outside thetransgene sequences, for example, 5′ and/or 3′ to the transgenesequences, for cleavage of the transgene. The nuclease cleavage site(s)may be for any nuclease(s). In certain embodiments, the nuclease targetsite(s) contained in the double-stranded donor are for the samenuclease(s) used to cleave the endogenous target into which the cleaveddonor is integrated via homology-independent methods.

The donor can be inserted so that its expression is driven by theendogenous promoter at the integration site, namely the promoter thatdrives expression of the endogenous gene into which the donor isinserted. However, it will be apparent that the donor may comprise apromoter and/or enhancer, for example a constitutive promoter or aninducible or tissue specific promoter. The donor molecule may beinserted into an endogenous gene such that all, some or none of theendogenous gene is expressed. Furthermore, although not required forexpression, exogenous sequences may also include transcriptional ortranslational regulatory sequences, for example, promoters, enhancers,insulators, internal ribosome entry sites, sequences encoding 2Apeptides and/or polyadenylation signals.

The transgenes carried on the donor sequences described herein may beisolated from plasmids, cells or other sources using standard techniquesknown in the art such as PCR. Donors for use can include varying typesof topology, including circular supercoiled, circular relaxed, linearand the like. Alternatively, they may be chemically synthesized usingstandard oligonucleotide synthesis techniques. In addition, donors maybe methylated or lack methylation. Donors may be in the form ofbacterial or yeast artificial chromosomes (BACs or YACs).

The donor polynucleotides described herein may include one or morenon-natural bases and/or backbones. In particular, insertion of a donormolecule with methylated cytosines may be carried out using the methodsdescribed herein to achieve a state of transcriptional quiescence in aregion of interest.

The exogenous (donor) polynucleotide may comprise any sequence ofinterest (exogenous sequence). Exemplary exogenous sequences include,but are not limited to any polypeptide coding sequence (e.g., cDNAs),promoter sequences, enhancer sequences, epitope tags, marker genes,cleavage enzyme recognition sites and various types of expressionconstructs. Marker genes include, but are not limited to, sequencesencoding proteins that mediate antibiotic resistance (e.g., ampicillinresistance, neomycin resistance, G418 resistance, puromycin resistance),sequences encoding colored or fluorescent or luminescent proteins (e.g.,green fluorescent protein, enhanced green fluorescent protein, redfluorescent protein, luciferase), and proteins which mediate enhancedcell growth and/or gene amplification (e.g., dihydrofolate reductase).Epitope tags include, for example, one or more copies of FLAG, His, myc,Tap, HA or any detectable amino acid sequence.

In some embodiments, the donor further comprises a polynucleotideencoding any polypeptide of which expression in the cell is desired,including, but not limited to antibodies, antigens, enzymes, receptors(cell surface or nuclear), hormones, lymphokines, cytokines, reporterpolypeptides, growth factors, and functional fragments of any of theabove. The coding sequences may be, for example, cDNAs.

In certain embodiments, the exogenous sequences can comprise a markergene (described above), allowing selection of cells that have undergonetargeted integration, and a linked sequence encoding an additionalfunctionality. Non-limiting examples of marker genes include GFP, drugselection marker(s) and the like.

In certain embodiments, the transgene may include, for example,wild-type genes to replace mutated endogenous sequences. For example, awild-type (or other functional) gene sequence may be inserted into thegenome of a stem cell in which the endogenous copy of the gene ismutated. The transgene may be inserted at the endogenous locus, or mayalternatively be targeted to a safe harbor locus.

Construction of such expression cassettes, following the teachings ofthe present specification, utilizes methodologies well known in the artof molecular biology (see, for example, Ausubel or Maniatis). Before useof the expression cassette to generate a transgenic animal, theresponsiveness of the expression cassette to the stress-inducerassociated with selected control elements can be tested by introducingthe expression cassette into a suitable cell line (e.g., primary cells,transformed cells, or immortalized cell lines).

Furthermore, although not required for expression, exogenous sequencesmay also transcriptional or translational regulatory sequences, forexample, promoters, enhancers, insulators, internal ribosome entrysites, sequences encoding 2A peptides and/or polyadenylation signals.Further, the control elements of the genes of interest can be operablylinked to reporter genes to create chimeric genes (e.g., reporterexpression cassettes). Exemplary splice acceptor site sequences areknown to those of skill in the art and include, by way of example only,CTGACCTCTTCTCTTCCTCCCACAG, (SEQ ID NO:42) (from the human HBB gene) andTTTCTCTCCACAG (SEQ ID NO:43) (from the human Immunoglobulin-gamma gene).

Targeted insertion of non-coding nucleic acid sequence may also beachieved. Sequences encoding antisense RNAs, RNAi, shRNAs and micro RNAs(miRNAs) may also be used for targeted insertions.

In additional embodiments, the donor nucleic acid may comprisenon-coding sequences that are specific target sites for additionalnuclease designs. Subsequently, additional nucleases may be expressed incells such that the original donor molecule is cleaved and modified byinsertion of another donor molecule of interest. In this way,reiterative integrations of donor molecules may be generated allowingfor trait stacking at a particular locus of interest or at a safe harborlocus.

Delivery

The nucleases, polynucleotides encoding these nucleases, donorpolynucleotides and compositions comprising the proteins and/orpolynucleotides described herein may be delivered by any suitable means.In certain embodiments, the nucleases and/or donors are delivered invivo. In other embodiments, the nucleases and/or donors are delivered toisolated cells (e.g., autologous or heterologous stem cells) for theprovision of modified cells useful in ex vivo delivery to patients.

Methods of delivering nucleases as described herein are described, forexample, in U.S. Pat. Nos. 7,888,121; 6,453,242; 6,503,717; 6,534,261;6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539;7,013,219; and 7,163,824, the disclosures of all of which areincorporated by reference herein in their entireties.

Nucleases and/or donor constructs as described herein may also bedelivered using any nucleic acid delivery mechanism, including naked DNAand/or RNA (e.g., mRNA) and vectors containing sequences encoding one ormore of the components. Any vector systems may be used including, butnot limited to, plasmid vectors, DNA minicircles, retroviral vectors,lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirusvectors and adeno-associated virus vectors, etc., and combinationsthereof. See, also, U.S. Pat. Nos. 6,534,261; 6,607,882; 6,824,978;6,933,113; 6,979,539; 7,013,219; and 7,163,824, and U.S. PatentPublication No. 20140335063 incorporated by reference herein in theirentireties. Furthermore, it will be apparent that any of these systemsmay comprise one or more of the sequences needed for treatment. Thus,when one or more nucleases and a donor construct are introduced into thecell, the nucleases and/or donor polynucleotide may be carried on thesame delivery system or on different delivery mechanisms. When multiplesystems are used, each delivery mechanism may comprise a sequenceencoding one or multiple nucleases and/or donor constructs (e.g., mRNAencoding one or more nucleases and/or mRNA or AAV carrying one or moredonor constructs).

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding nucleases and donor constructs incells (e.g., mammalian cells) and target tissues. Non-viral vectordelivery systems include DNA plasmids, DNA minicircles, naked nucleicacid, and nucleic acid complexed with a delivery vehicle such as aliposome or poloxamer. Viral vector delivery systems include DNA and RNAviruses, which have either episomal or integrated genomes after deliveryto the cell.

Methods of non-viral delivery of nucleic acids include electroporation,lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, nanoparticles, polycation or lipid:nucleic acidconjugates, naked DNA, naked RNA, capped RNA, artificial virions, andagent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron2000 system (Rich-Mar) can also be used for delivery of nucleic acids.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) andCopernicus Therapeutics Inc, (see for example U.S. Pat. No. 6,008,336).Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787;and 4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Felgner, WO 91/17424, WO 91/16024.

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered CRISPR/Cas systems take advantage of highlyevolved processes for targeting a virus to specific cells in the bodyand trafficking the viral payload to the nucleus. Viral vectors can beadministered directly to subjects (in vivo) or they can be used to treatcells in vitro and the modified cells are administered to subjects (exvivo). Conventional viral based systems for the delivery of CRISPR/Cassystems include, but are not limited to, retroviral, lentivirus,adenoviral, adeno-associated, vaccinia and herpes simplex virus vectorsfor gene transfer. Integration in the host genome is possible with theretrovirus, lentivirus, and adeno-associated virus gene transfermethods, often resulting in long term expression of the insertedtransgene. Additionally, high transduction efficiencies have beenobserved in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof.

In applications in which transient expression is preferred, adenoviralbased systems can be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and high levels of expressionhave been obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors arealso used to transduce cells with target nucleic acids, e.g., in the invitro production of nucleic acids and peptides, and for in vivo and exvivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47(1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994).Construction of recombinant AAV vectors are described in a number ofpublications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol.Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989). Any AAV serotype can beused, including AAV1, AAV3, AAV4, AAV5, AAV6 and AAV8, AAV 8.2, AAV9,and AAV rh10 and pseudotyped AAV such as AAV2/8, AAV2/5 and AAV2/6.

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138(1997)). PA317/pLASN was the first therapeutic vector used in a genetherapy trial. (Blaese et al., Science 270:475-480 (1995)). Transductionefficiencies of 50% or greater have been observed for MFG-S packagedvectors. (Ellem et al., Immunol Immunother 44(1):10-20 (1997); Dranoffet al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 base pair (bp)inverted terminal repeats flanking the transgene expression cassette.Efficient gene transfer and stable transgene delivery due to integrationinto the genomes of the transduced cell are key features for this vectorsystem. (Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al.,Gene Ther. 9:748-55 (1996)). Other AAV serotypes, including AAV1, AAV3,AAV4, AAV5, AAV6, AAV8, AAV9 and AAVrh10, and all variants thereof, canalso be used in accordance with the present invention.

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including non-dividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for anti-tumorimmunization with intramuscular injection (Sterman et al., Hum. GeneTher. 7:1083-9 (1998)). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et al.,Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:71083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarezet al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther.5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. USA 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual subject, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingnucleases and/or donor constructs can also be administered directly toan organism for transduction of cells in vivo. Alternatively, naked DNAcan be administered. Administration is by any of the routes normallyused for introducing a molecule into ultimate contact with blood ortissue cells including, but not limited to, injection, infusion, topicalapplication and electroporation. Suitable methods of administering suchnucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Vectors suitable for introduction of polynucleotides described hereininclude non-integrating lentivirus vectors (IDLV). See, for example, Oryet al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al.(1998) J. Virol. 72:8463-8471; Zuffery et al. (1998) J. Virol.72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222; U.S.Patent Publication No 2009/054985.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

It will be apparent that the nuclease-encoding sequences and donorconstructs can be delivered using the same or different systems. Forexample, a donor polynucleotide can be carried by an AAV, while the oneor more nucleases can be carried by mRNA. Furthermore, the differentsystems can be administered by the same or different routes(intramuscular injection, tail vein injection, other intravenousinjection, intraperitoneal administration and/or intramuscularinjection. The vectors can be delivered simultaneously or in anysequential order.

Formulations for both ex vivo and in vivo administrations includesuspensions in liquid or emulsified liquids. The active ingredientsoften are mixed with excipients which are pharmaceutically acceptableand compatible with the active ingredient. Suitable excipients include,for example, water, saline, dextrose, glycerol, ethanol or the like, andcombinations thereof. In addition, the composition may contain minoramounts of auxiliary substances, such as, wetting or emulsifying agents,pH buffering agents, stabilizing agents or other reagents that enhancethe effectiveness of the pharmaceutical composition.

Kits

Also provided are kits comprising any of the linkers described hereinand/or for performing any of the above methods. The kits typicallycontain a linker sequence as described herein (or a polynucleotideencoding a linker as described herein). The kit may supply the linkeralone or may provide vectors into which a DNA-binding domain and/ornuclease of choice can be readily inserted into. The kits can alsocontain cells, buffers for transformation of cells, culture media forcells, and/or buffers for performing assays. Typically, the kits alsocontain a label which includes any material such as instructions,packaging or advertising leaflet that is attached to or otherwiseaccompanies the other components of the kit.

Applications

The disclosed linkers are advantageously used to link with engineeredDNA-binding domains with cleavage domains to form nucleases for cleavingDNA. The linkers as described herein allow for the cleavage of DNA whenthe target sites of a pair of nucleases used for cleavage are ofvariable spacings, for example target sites that are not 5 or 6 basepairs apart (e.g., 7, 8, 9 or more base pairs apart). Cleavage can be ata region of interest in cellular chromatin (e.g., at a desired orpredetermined site in a genome, for example, in a gene, either mutant orwild-type); to replace a genomic sequence (e.g., a region of interest incellular chromatin) with a homologous non-identical sequence (i.e.,targeted recombination); to delete a genomic sequence by cleaving DNA atone or more sites in the genome, which cleavage sites are then joined bynon-homologous end joining (NHEJ); to screen for cellular factors thatfacilitate homologous recombination; and/or to replace a wild-typesequence with a mutant sequence, or to convert one allele to a differentallele. Such methods are described in detail, for example, in U.S. Pat.No. 7,888,121, incorporated by reference in its entirety herein.

Accordingly, the disclosed linkers can be used in any nuclease for anymethod in which specifically targeted cleavage is desirable and/or toreplace any genomic sequence with a homologous, non-identical sequence.For example, a mutant genomic sequence can be replaced by its wild-typecounterpart, thereby providing methods for treatment of e.g., geneticdisease, inherited disorders, cancer, and autoimmune disease. In likefashion, one allele of a gene can be replaced by a different alleleusing the methods of targeted recombination disclosed herein. Indeed,any pathology dependent upon a particular genomic sequence, in anyfashion, can be corrected or alleviated using the methods andcompositions disclosed herein.

Exemplary genetic diseases include, but are not limited to,achondroplasia, achromatopsia, acid maltase deficiency, adenosinedeaminase deficiency (OMIM No. 102700), adrenoleukodystrophy, aicardisyndrome, alpha-1 antitrypsin deficiency, alpha-thalassemia, Alsheimer'sdisease, androgen insensitivity syndrome, apert syndrome, arrhythmogenicright ventricular, dysplasia, ataxia telangictasia, barth syndrome,beta-thalassemia, blue rubber bleb nevus syndrome, canavan disease,chronic granulomatous diseases (CGD), cri du chat syndrome, cysticfibrosis, dercum's disease, ectodermal dysplasia, fanconi anemia,fibrodysplasia ossificans progressive, fragile X syndrome, galactosemis,Gaucher's disease, generalized gangliosidoses (e.g., GM1),hemochromatosis, the hemoglobin C mutation in the 6^(th) codon ofbeta-globin (HbC), hemophilia, Huntington's disease, Hurler Syndrome,hypophosphatasia, Klinefleter syndrome, Krabbes Disease, Langer-GiedionSyndrome, leukocyte adhesion deficiency (LAD, OMIM No. 116920),leukodystrophy, long QT syndrome, Marfan syndrome, Moebius syndrome,mucopolysaccharidosis (MPS), nail patella syndrome, nephrogenic diabetesinsipdius, neurofibromatosis, Neimann-Pick disease, osteogenesisimperfecta, Parkinson's disease, porphyria, Prader-Willi syndrome,progeria, Proteus syndrome, retinoblastoma, Rett syndrome,Rubinstein-Taybi syndrome, Sanfilippo syndrome, severe combinedimmunodeficiency (SCID), Shwachman syndrome, sickle cell disease (sicklecell anemia), Smith-Magenis syndrome, Stickler syndrome, Tay-Sachsdisease, Thrombocytopenia Absent Radius (TAR) syndrome, Treacher Collinssyndrome, trisomy, tuberous sclerosis, Turner's syndrome, urea cycledisorder, von Hippel-Landau disease, Waardenburg syndrome, Williamssyndrome, Wilson's disease, Wiskott-Aldrich syndrome, X-linkedlymphoproliferative syndrome (XLP, OMIM No. 308240) and X-linked SCID.

Additional exemplary diseases that can be treated by targeted DNAcleavage and/or homologous recombination include acquiredimmunodeficiencies, lysosomal storage diseases (e.g., Gaucher's disease,GM1, Fabry disease and Tay-Sachs disease), mucopolysaccahidosis (e.g.Hunter's disease, Hurler's disease), hemoglobinopathies (e.g., sicklecell diseases, HbC, α-thalassemia, β-thalassemia) and hemophilias.

Targeted cleavage of infecting or integrated viral genomes can be usedto treat viral infections in a host. Additionally, targeted cleavage ofgenes encoding receptors for viruses can be used to block expression ofsuch receptors, thereby preventing viral infection and/or viral spreadin a host organism. Targeted mutagenesis of genes encoding viralreceptors (e.g., the CCR5 and CXCR4 receptors for HIV) can be used torender the receptors unable to bind to virus, thereby preventing newinfection and blocking the spread of existing infections. See,International Patent Publication WO 2007/139982. Non-limiting examplesof viruses or viral receptors that may be targeted include herpessimplex virus (HSV), such as HSV-1 and HSV-2, varicella zoster virus(VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV), HHV6 andHHV7. The hepatitis family of viruses includes hepatitis A virus (HAV),hepatitis B virus (HBV), hepatitis C virus (HCV), the delta hepatitisvirus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV). Otherviruses or their receptors may be targeted, including, but not limitedto, Picornaviridae (e.g., polioviruses, etc.); Caliciviridae;Togaviridae (e.g., rubella virus, dengue virus, etc.); Flaviviridae;Coronaviridae; Reoviridae; Birnaviridae; Rhabodoviridae (e.g., rabiesvirus, etc.); Filoviridae; Paramyxoviridae (e.g., mumps virus, measlesvirus, respiratory syncytial virus, etc.); Orthomyxoviridae (e.g.,influenza virus types A, B and C, etc.); Bunyaviridae; Arenaviridae;Retroviradae; lentiviruses (e.g., HTLV-I; HTLV-II; HIV-1 (also known asHTLV-III, LAV, ARV, hTLR, etc.) HIV-II); simian immunodeficiency virus(SIV), human papillomavirus (HPV), influenza virus and the tick-borneencephalitis viruses. See, e.g. Virology, 3rd Edition (W. K. Joklik ed.1988); Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe,eds. 1991), for a description of these and other viruses. Receptors forHIV, for example, include CCR-5 and CXCR-4.

Nucleases containing the disclosed linkers can also be used forinactivation (partial or complete) of one or more genomic sequences.Inactivation can be achieved, for example, by a single cleavage event,by cleavage followed by non-homologous end joining, by cleavage at twosites followed by joining so as to delete the sequence between the twocleavage sites, by targeted recombination of a missense or nonsensecodon into the coding region, by targeted recombination of an irrelevantsequence (i.e., a “stuffer” sequence) into the gene or its regulatoryregion, so as to disrupt the gene or regulatory region, or by targetingrecombination of a splice acceptor sequence into an intron to causemis-splicing of the transcript.

Nuclease-mediated inactivation (e.g., knockout) of endogenous genes canbe used, for example, to generate cell lines deficient in genes involvedin apoptosis or protein production (e.g., post-translationalmodifications such as fucosylation). ZFN-mediated inactivation can alsobe used to generate transgenic organisms (e.g., plants, rodents andrabbits).

In addition, because nucleases don't appear to have specificity for theDNA sequence between the two paired half sites, nucleases with linkersas described herein can be designed to cleave DNA such that theresulting single-stranded overhangs have any desired sequence. Inparticular, linkers as described herein can be designed to influenceboth the size and position of these single-stranded overhangs withrespect to the starting sequence. Thus, when incorporated into one ormore nucleases of a nuclease pair, linkers as described herein canresult in more uniform ends following cleavage. Accordingly, the linkersdescribed herein can also be used to more efficiently clone DNA cut withnucleases, which is broadly applicable in many areas of biotechnologyand basic science.

Thus, the linkers described herein provide broad utility for improvingnuclease-mediated cleavage in gene modification applications. Linkers asdescribed herein may be readily incorporated into any existing nucleaseby either site directed mutagenesis or subcloning to be used in manyapplications in standard cloning, constructing large genomes forsynthetic biology, new types of RFLP analysis of large sequences or evenallow new types of cloning involving extremely large DNA sequences. Thepotential properties of nucleases with rigid linkers could also be idealin applications such as DNA computing.

The following Examples relate to exemplary embodiments of the presentdisclosure in which the nuclease comprises one or more ZFNs. It will beappreciated that this is for purposes of exemplification only and thatother nucleases can be used, for instance TALENs, homing endonucleases(meganucleases) with engineered DNA-binding domains and/or fusions ofnaturally occurring of engineered homing endonucleases (meganucleases)DNA-binding domains and heterologous cleavage domains, and nucleasesystems such as TtAgo and CRISPR/Cas using engineered single guide RNAs.

EXAMPLES Example 1: Design and Construction of ZFNs with Rigid Linkers

Zinc finger nuclease constructs targeted to the human CCR5 locus wereprepared as disclosed in U.S. Pat. No. 7,951,925. “Wild-type constructs”included the “ZC” linker.

In addition, pairs of ZFNs targeted to sequences in the humanmitochondria containing the mutation that causes MELAS (mitochondrialmyopathy, encephalopathy, lactic acidosis, and stroke) were alsoprepared to include the L7a or L6a linker as described in U.S. PatentPublication No. 20090305419.

Example 2: ZFN Activity

A. CCR5-Targeted ZFNs

Constructs encoding CCR5-targeted ZFN SBS #8266 were initially tested ina yeast Mel-I reporter system as described in U.S. Pat. No. 8,563,314.In particular, yeast strains having an inverted repeat of the SBS #8266target site separated by 3, 4, 5, 6, 7, or 8 bp were used tocharacterize the constructs.

The wild-type ZFN (with the standard LRGSQLVKSELEEKKS (SEQ ID NO:44)linker showed strong activity with 5 bp and 6 bp half site spacings. Inaddition, the constructs with the L6a linker sequence (FIG. 2) showedactivity at 6p spacings and the L7a linker sequence showed significantactivity with 7 bp and 8 bp spacings.

In vitro DNA binding and cleavage activity of the MELAS-targeted ZFNswas also assayed and pairs of ZFNs including the rigid L7a linkercleaved their target.

Finally, the CCR5 ZFNs including the L7a linker were tested for NHEJactivity at the endogenous human CCR5 locus in cell lines that containvarious numbers of base pairs between the half sites. Results are shownin Table 1.

TABLE 1 target sites % NHEJ % NHEJ % NHEJ ZFN separated by (exp't #1)(exp't #2) (average) Wt ZFNs 4 bp 1.2 1.1 1.2 Wt ZFNs 5 bp 36.0 34.035.0 Wt ZFNs 6 bp 13.4 8.8 11.1 Wt ZFNs 7 bp 0.0 0.0 0.0 Wt ZFNs 8 bp0.0 0.0 0.0 L6a ZFNs 4 bp 0.0 0.0 0.0 L6a ZFNs 5 bp 44.4 34.1 39.3 L6aZFNs 6 bp 26.2 24.6 25.4 L6a ZFNs 7 bp 6.5 3.7 5.1 L6a ZFNs 8 bp 0.0 0.00.0 L7a ZFNs 4 bp 0.0 0.0 0.0 L7a ZFNs 5 bp 0.0 0.0 0.0 L7a ZFNs 6 bp33.1 30.5 31.8 L7a ZFNs 7 bp 41.1 38.1 39.6 L7a ZFNs 8 bp 7.9 4.6 6.1

As expected, the wild-type ZFNs only showed high activity at half-sitesseparated by 5 or 6 bp. However, CCR5-targeted ZFNs including the rigidL7a linker showed high activity with a 7 bp spacing and noticeableactivity with the 8 bp spacing. It should be noted that the efficiencyof the L7a constructs with the 7 bp spacing is very similar to theefficiency of the wild type ZFNs with a 5 bp spacing (either in thewild-type cell line or a cell line with a different sequence of the 5 bpin between the half sites).

In addition, combinations of linkers were also tested in CCR5-targetedZFN pairs. Briefly, K562 cells were engineered to have gaps of 4 to 8base pairs (bp) between the CCR5 ZFN binding sites. Two CCR5 ZFNs withdifferent linkers combinations (Wt/L7a) were transfected into these K562cells by Amaxa Shuttle. Samples were harvested 3 days after transfectionand subjected to CEL1-I assay analysis. CEL-I mismatch assays wereperformed essentially as per the manufacturer's instructions(Trangenomic SURVEYOR™).

The results indicate that the Wt/Wt linker ZFN has the highest activitywith 5 bp gap target sequence; the L7a/L7a linker ZFN had the highestactivity with a 7 bp gap sequence, and the ZFNs with Wt/L7a or L7a/Wtlinker combinations had the highest activity with a 6 bp gap sequence.

B. ROSA-Targeted ZFNs

Neuro2A cells were transfected with combinations of mROSA-targeted ZFNs(see, e.g., U.S. Patent Publication No. 2007/0134796) by Amaxa Shuttleusing a target site with a 6 bp gap. One ZFN of the pairs included awild-type linker (“ZC”) and the other included either wild-type or L7alinker as described herein. Samples were harvested 3 days aftertransfection and subjected to CEL-I analysis, as described above.

As shown in Table 2 below, the wild type (WT)/L7a linker in a pair ofZFNs is active with a 6 bp gap.

TABLE 2 Sample Linker #1 Linker #2 % NHEJ mock transfection (no ZFN) NANA 0.4 Rosa-ZFN pairs Wt Wt 22.6 Rosa-ZFN pairs Wt L7a 7.5 Rosa-ZFNpairs Wt Wt 23.2 Rosa-ZFN pairs Wt Wt 18.7 GFP-ZFN pairs Wt L7a 5.3GFP-ZFN pairs Wt Wt 21.5C. Rat IgM

Rat C6 cells were transfected with combinations of rat IgM-targeted ZFNs(see, e.g., U.S. Publication No. 20100218264) by Amaxa Shuttle using atarget site with a 6 bp gap. One ZFN of the pairs included a wild-typelinker (“ZC”) and the other included either wild-type or L7a linker.Samples were harvested 9 days after transfection and subjected to CEL-Ianalysis, as described above and in U.S. Patent Publication No.2007/0134796.

Cells containing the pair of ZFNs that included the L7a linkers showed2.43% NHEJ as compared to cells containing a pair of ZFNs that includedthe ZC linker, which showed 1.93% NHEJ. Furthermore, the L7a-containinglinker ZFN pair was used to inject into rat ES cells (as described inU.S. Publication No. 20100218264) and these ES cells successfullyproduced homozygous IgM gene knockout rat offspring.

Example 3: Additional Linker Designs

Additional linkers were generated from a bacterial selection system thatwas modified from Barbas et al. (2010) J. Mol. Biol. 400:96-107.Briefly, bacteria were transformed with a ZFN-encoding plasmid(expression of the ZFN driven by the arabinose inducible promoter) and aplasmid that expressed the bacterial ccdB toxin from the T7 promoter andincluded the ZFN target sites. See, FIG. 2. This system allowed theability to query very large libraries with complexities of ˜10⁸ and ahigh stringency option (>100 cleavage events required for survival).

The expressed ZFN cleaved pTox leading to degradation of pTox; the ccdBtoxin was then switched on for cell killing; the survivors (ZFN-cleavedpTox) were amplified and the genes encoding the linkers re-cloned intonew plasmids for the next cycle. See, FIGS. 2A and 2B. The ZFNs includedfully randomized linkers of between 8 and 17 amino acids in lengthbetween the zinc finger protein domain and the cleavage (FokI) domain.See, FIG. 2A.

Engineered cleavage domains and canonical (C2H2) and non-canonicalfinger structures (see, U.S. Patent Publication No. 20080182332) may beused; for this study wild-type FokI domains (which form homodimers) andCCHC finger structures were employed. Linkers were selected on gapsbetween ZFN target sequences of 5-16 base pairs.

To select for active pZFNs from the vast excess of inactive ones, thefollowing steps were performed: (i) High-complexity DNA libraries wereconstructed, see FIG. 2A, (ii) plasmid DNA library was transformed intocells bearing pTox_8196-bs; (iii) expressed ZFNs for 2 hours; (iv)induced ccdB overnight; (v) minipreped plasmids and subclone linker DNAinto new pZFN; repeated (ii)-(v) for 7 more cycles.

Exemplary functional linkers obtained from the selections are shown inFIG. 4. A summary of the length distribution of the linkers whichcleaved at the indicated gaps is shown in FIG. 5.

Example 4: Verifying Activity and Portability Studies

Selected candidates (ZFNs with linkers) were screened for modificationof an endogenous target in six variant K562 cell lines, which wereengineered at the CCR5 locus to replace the gap between heterodimerictarget sites (8166 and 8266 ZFNs as described in U.S. Pat. No.7,951,925) with new gaps of 5-16 base pairs (e.g., 5, 6, 7, 8, 15 or 16bp). Linker cassettes (˜80/selection=480 total) were substituted for L0linkers in CCR5 ZFN expression vectors with the CCHC structure andconstructs were transfected into appropriate K562 cells. See, FIG. 6.Activity was assessed using a Cel1 assay, as described above and in U.S.Patent Publication No. 2007/0134796.

FIG. 8 shows a summary of the results obtained with the selected linkerswith the indicated target gaps. FIGS. 9A and 9B show the most activelinkers for a 5-8 base pair gap between target sites. “Indels” refers toinsertions or deletions, usually small, within the target sequencefollowing ZFN-mediated modification (e.g., following cleavage and NHEJrepair). Linkers were also tested for activity with different gapspacings. FIG. 9C shows the gap preference for the indicated linkers.

For portability studies, four L8 linkers were chosen for cloning andtesting with a large target gene to ensure consistent high design scoresfor all test sets. A schematic of the vector designs is shown in FIG.10A. Sets of the 32 ZFNs were generated for each of L0 (5, 6 bp gap),L7a (7 bp gap), and L8 (8 bp gap) linkers for a total of 92 ZFNs. See,FIG. 7 for L8 linkers. Activity was verified by Cel-1 assay, asdescribed above and in U.S. Patent Publication No. 2007/0134796.

FIG. 10B shows a summary of the results of the portability studies anddemonstrates many ZFNs tested were active. The number active and levelof modification of the selected linkers was comparable to previouslyused linkers at a 5 or 6 (L0) or 7 bp spacing (L7a).

The L8c linkers were also additionally modified. A different target waschosen and eight pairs of C2H2 ZFNs were used to select for activity for8 bp (termed A, B, C, D) and 9 bp (termed E, F, G, and H) gaps againsttheir specific targets. The linkers in the ZFN pairs were altered with 4different linker types (termed L8n, L8o, L8m and L8p) and the pairs wereselected for activity against target sites where the gaps between theZFN binding sites were separated by 8 or 9 bp. Additionally, theoriginal L8c linker was used in the pairs to attach either a wt Fok1nuclease domain or enhanced obligated heterodimeric Fok1 nucleasedomains (eHiFi) for comparison. A GFP expression vector was used as atransfection control. The peptide sequences of the original L8c linkerand the 4 modified linkers are shown below (linker sequencesunderlined):

Group 1: L8c (SEQ ID NO: 45)[ZFP]HAQRC-NGSYAPMPPLALASPELEEKESEL[wt FokI] Group 2: L8c(SEQ ID NO: 45) [ZFP]HAQRC-NGSYAPMPPLALASPELEEKESEL[eHiFi FokI]Group 3: L8n (SEQ ID NO: 46)[ZFP]HTKIH-NGSYAPMPPLALASPELEEKESEL[eHiFi FokI] Group 4: L8o(SEQ ID NO: 47) [ZFP]HTKIH-GGSYAPMPPLALASPELEEKESEL[eHiFi FokI]Group 5: L8m (SEQ ID NO: 48)[ZFP]HTKIH--GSYAPMPPLALASPELEEKESEL[eHiFi FokI] Group 6: L8p(SEQ ID NO: 49) [ZFP]HTKIHLRGSYAPMPPLALASPELEEKESEL[eHiFi FokI]

The results (see FIGS. 11A and 11B) demonstrated that the L8c typelinker worked well in both the CCHC and C2H2 ZFP backgrounds.Additionally, the L8p linker worked well with both 8 bp and 9 bp gaps.

Example 5: Further Linker Studies

The “L7a” linker is currently used in as the ZFP-FokI linker to targetthe 7-bp gap sites in both C2H2 and CCHC ZFP architectures. Linkersshown in FIGS. 9A and 9B were identified within a CCHC ZFP architectureso further studies were conducted in C2H2 architecture of ZFPs thattarget 7-bp gap sites.

Four linker peptide sequences as shown in FIG. 9A (L7-1, L7-3, L8a, andL8c) were initially selected for these studies, including these linkerswith and without one or more optional residues as shown in parenthesesas follows: (H)LPKPANPFPLD (SEQ ID NO:7); (D)PNSPISRARPLNPHP (SEQ IDNO:10); (N)GICPPPRPRTSPP (SEQ ID NO:2); and (N)GSYAPMPPLALASP (SEQ IDNO:4).

A pair of ZFPs that targets a 7-bp gap site was used to test thelinkers. This pair of ZFNs was engineered in the context of CCHC or C2H2architectures, with various linker sequences, and fused to eitherwild-type (“wt”) or enhanced obligated heterodimeric FokI (“eHiFi”)domains. See, e.g., U.S. Pat. Nos. 8,623,618; 7,888,121; 7,914,796; and8,034,598 and U.S. Publication No. 20110201055 for exemplary eHiFidomains. These ZFN pairs were tested in K562 cells and their nucleasecleavage activities were measured by Cel1 assay and MiSeq sequencing.

The results, as shown in FIG. 12, demonstrated that (1) these linkersare effective in both wild type and eHiFi FokI architectures (comparingGroup 1 and Group 2); (2) these linkers are effective in the context ofboth C3H and C2H2 architecture (comparing Group 2 and Group 3); and (3)removing one amino acid residue (from the N-terminal) in each linker(shown in Group 3) resulted in significantly higher activity as comparedto linkers with the C-terminal residue and, in most instance, ascompared to the currently-used L7a linker (sample #17 in FIG. 12).

Subsequently, the two most active 7-bp linker sequences (“L7c3” and“L8c3”, indicated by arrows) were modified to make the C-terminus of aZFP ends with the conventional “HTKIHLRGS” peptide sequence (SEQ ID NO:50), which allows ZFN assembly using the same process as all other C2H2ZFNs.

To ensure the modified linkers possessed the same or higher activitiesthan the conventional “L7a” linker on a 7-bp gap site, four linkervariants were designed for both “L7c3” and “L8c3” and engineered intotwo ZFN pairs (see FIG. 13). These ZFNs were tested in human CD34 cellsand their NHEJ activities were measured with MiSeq analyses.

As shown in FIG. 13, while all variants tested were active, many of themshowed higher activities than the conventional “L7a” bearing ZFNs,including the highest activity as seen with “L7c5” and “L7e4” variants.

Example 6: Optimizing ZFN Activity by Changing the ZFP-FokI JunctionSequences

The conventional junction sequence between ZFP and FokI nuclease domainis “-HTKIHLRGSQLVKSELEEK-” (SEQ ID NO:51). Since ZFN activity can beaffected by many factors including the gap length between the two ZFPbinding sites and the ZFP affinity to its binding sites, we testedwhether the cleavage efficiency of a pair of ZFNs could be modulated bychanging the length and/or compositions at the junction sites tooptimize the heterodimer interaction between the two FokI cleavagedomains. See, FIG. 14. ZFNs of C2H2 or C3H structure can be used.

In particular, a series of 24 junction variants were designed andengineered into a pair of ZFNs target to a 6-bp gap site. See, FIG. 15.Some junction variants have the same length as the conventional one butwith different (substituted) amino acid residue(s). In addition, thesevariants also contain additional amino acids or have some amino acidsremoved (as shown in FIG. 15).

These ZFNs were tested in K562 cells in the following ways: (1) the 24left ZFN variants were paired with the right ZFN containing theconventional linker, (b) the 24 right ZFN variants were paired with theleft ZFN containing the conventional linker, and (c) the 24 left ZFNvariants were paired with each of the right ZFN variants. The ZFN NHEJactivities were measured by MiSeq.

As shown in FIG. 15, ZFN activities were affected by pairing ZFNscontaining the various junction linkers. Notably, many of the modifiedjunction regions showed significantly higher NHEJ activities whencomparing to the pair of ZFNs containing both of the conventionaljunction sequences.

Selected junction sequences were also tested using ZFN pairs that targeta different site. Two ZFNs pairs with the junction variants weretransfected into human HepG2 cells and their NHEJ activities weremeasured by MiSeq. The data as shown in FIG. 16 demonstrated again thatthe ZFN activities can be further improved by modulating the ZFP-FokIjunction sequences.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entirety.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

What is claimed is:
 1. A fusion protein comprising a DNA-binding domainhaving an N-terminus and a C-terminus, wherein the DNA-binding domainbinds to a nucleotide target site; a truncated FokI cleavage domain inwhich the N-terminal residues QLVKS (residues 158 to 162 of SEQ ID NO:1)are deleted; and a linker between the C-terminus of the DNA-bindingdomain and the N-terminus of the cleavage domain, wherein the linkercomprises a sequence selected from the group consisting of(N)GICPPPRPRTSPP (L8a, SEQ ID NO:2); (T)GTAPIEIPPEVYP (L8b, SEQ IDNO:3); (N)GSYAPMPPLALASP (L8c, SEQ ID NO:4); (P)GIYTAPTSRPTVPP (L8d, SEQID NO:5); (N)GSQTPKRFQPTHPSA (L8e, SEQ ID NO:6); TGLMPPSHPRQPIHINF (L8g,SEQ ID NO:234); TGTVHTSPICPQTYP (L8i, SEQ ID NO:161); TGSGTPTRPHPPLPP(L8j, SEQ ID NO:235); (H)LPKPANPFPLD (L7-1, SEQ ID NO:7);(H)RDGPRNLPPTSPP (L7-2, SEQ ID NO:8); (H)RLPDSPTALAPDTL (L7-6, SEQ IDNO:9); (D)PNSPISRARPLNPHP (L7-3, SEQ ID NO:10); (Y)GPRPTPRLRCPIDSLIFR(L7-5, SEQ ID NO:11); (H)CPASRPIHP (L6-2, SEQ ID NO:12); (G)LQSLIPQQLL(L6-6, SEQ ID NO:13); (G)LQPTVNHEYNN (L6-7, SEQ ID NO:14); and(P)ANIHSLSSPPPL (L6-1, SEQ ID NO:15) and further wherein the amino acidresidue shown in round parentheses is optionally present.
 2. The fusionprotein of claim 1, wherein the linker further comprises a ZC sequence(SEQ ID NO:35).
 3. The fusion protein of claim 2, wherein the ZCsequence is at the N-terminus of the linker.
 4. The fusion protein ofclaim 1, wherein the DNA-binding domain is a zinc finger protein.
 5. Adimer comprising two fusion proteins according to claim
 1. 6. The dimerof claim 5, wherein the dimer is a homodimer or heterodimer.
 7. Apolynucleotide encoding at least one fusion protein according toclaim
 1. 8. A cell comprising a fusion protein according to claim
 1. 9.A cell comprising a polynucleotide according to claim
 7. 10. A methodfor targeted cleavage of cellular chromatin in a region of interest in acell, the method comprising: expressing a pair of nucleases in the cellunder conditions such that cellular chromatin is cleaved at the regionof interest, wherein the nucleases bind to target sites in the region ofinterest and further wherein at least one nuclease comprises a fusionprotein according to claim
 1. 11. The method of claim 10, wherein bothnucleases comprise fusions proteins according to claim
 1. 12. The methodof claim 10, wherein the target sites for the zinc finger nucleases are3 to 20 base pairs apart.
 13. The method of claim 10, further comprisingthe step of introducing a donor polynucleotide into the cell, whereinall or part of the donor polynucleotide is incorporated into the regionof interest following cleavage.
 14. A kit for producing a nuclease, thekit comprising a fusion protein according to claim 1 contained in one ormore containers, optional hardware, and instructions for use of the kit.15. A kit for producing a nuclease, the kit comprising a polynucleotideaccording to claim 7 contained in one or more containers, optionalhardware, and instructions for use of the kit.
 16. The kit of claim 14,further comprising a donor polynucleotide.